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Definitions

• Lysine specific histone demethylase-1 inhibitors and uses therefor
• This invention relates generally to lysine specific histone demethylase-1 (LSD1) inhibitors in methods and compositions for immune checkpoint inhibition.
• the invention also relates to proteinaceous molecules and their use in altering at least one of (i) formation, (ii) proliferation, (iii) maintenance, (iv) epithelial to mesenchymal cell transition (EMT), or (v) mesenchymal to epithelial cell transition (M ET) of an LSD1 overexpressing cell.
• PD-1 Programmed cell death protein-1 plays an important role in regulation of the immune system through its ability to regulate T cell activation and reduce the immune response.
• PD-1 is expressed on activated T cells (including immunosuppressive CD4+ T cells (Treg) and exhausted CD8+ T cells), B cells, myeloid dendritic cells (MDCs), monocytes, thymocytes and natural killer (NK) cells (Gianchecchi er a/. (2013) Autoimmun. Rev. , 12: 1091-1100).
• the PD-1 signaling pathway contributes to the maintenance of central and peripheral tolerance in normal individuals, thereby avoiding destruction of normal host tissue.
• the interaction of PD-1 and its ligands suppresses positive selection, thereby inhibiting the transformation of CD4- CD8- double negative cells to CD4+ CD8+ double positive T cells (Keir ef a/. (2005) J. Immunol., 175 : 7329-7379).
• Inhibition of self-reactive and inflammatory effector T cells that escape negative selection to avoid collateral immune-mediated tissue damage is dependent on the PD-1 signaling pathway (Keir er a/. (2006) J. Exp. Med. , 203 : 883-895).
• PD-1 is bound by two ligands: programmed cell death ligand-1 (PD-L1 ; B7-H1 ; CD274) and programmed cell death ligand-2 (PD-L2; B7-DC; CD273).
• PD-L1 is expressed on various cell types, including T cells, B cells, dendritic cells, macrophages, epithelial cells and endothelial cells (Chen er a/. (2012) Clin Cancer Res, 18(24) : 6580-6587; Herzberg er a/. (2016) The Oncologist, 21 : 1-8).
• PD-L1 expression is also upregulated in many types of tumor cells and other cells in the local tumor environment (Herzberg er a/.
• PD-L2 is predominantly expressed on antigen-presenting cells such as monocytes, macrophages and dendritic cells, but expression may also be induced on a wide variety of other immune cells and non-immune cells depending on microenvironmental stimuli (Herzberg er a/. (2016) The Oncologist, 21 : 1-8; Kinter er a/. (2008) J. Immunol. , 181 : 6738-6746; Zhong er a/. (2007) Eur. J. Immunol. , 37 : 2405- 2410; Messal er a/. (2011) Mol. Immunol. , 48: 2214-2219; Lesterhuis er a/. (2011) Mol.
• PD-1, PD-L1 and PD-L2 are overexpressed by malignant cells and other cells in the local tumor environment.
• PD-1 is highly expressed on a large proportion of tumor-infiltrating lymphocytes (TILs) from many different tumor types and suppresses local effector immune responses.
• TILs tumor-infiltrating lymphocytes
• TIL expression of PD-1 is associated with impaired effector function (cytokine production and cytotoxic efficacy against tumor cells) and/or poor outcome in numerous tumor types (Thompson et al. (2007) Clin Cancer Res, 13(6) : 1757-1761 ; Shi et al. (2011) Int. J. Cancer, 128 : 887-896).
• PD-L1 expression has been found to strongly correlate with poor outcome in many tumor types, including kidney, ovarian, bladder, breast, urothelial, gastric and pancreatic cancer (Keir et al. (2008) Annu. Rev. Immunol. , 26: 677-704; Shi et al. (2011) Int. J. Cancer, 128 : 887- 896).
• PD-L2 has been shown to be upregulated in a subset of tumors and has also been linked to poor outcome.
• members of the PD-1 signaling pathway are important therapeutic targets for the treatment of cancer and infection and new therapeutic agents targeting this pathway are desired.
• LSDl inhibitors inhibit immune checkpoints, particularly PD-L1 and/or PD-L2. Accordingly, the inventors have conceived that LSDl inhibitors may be used for a range of applications, including for enhancing an immune response in a subject to a target antigen by an immune-modulating agent or for the treatment of a cancer, particularly a metastatic cancer, or an infection.
• a LSDl inhibitor for inhibiting PD-L1 and/or PD-L2 activity in a subject.
• a method of inhibiting PD-L1 and/or PD-L2 activity in a subject comprising administering an LSDl inhibitor to the subject.
• a method of inhibiting the nuclear translocation of PD-L1 and/or PD-L2 in subject comprising administering an LSDl inhibitor to the subject.
• an LSDl inhibitor for enhancing an immune response in a subject to a target antigen by an immune-modulating agent.
• the present invention provides a composition comprising an LSDl inhibitor and an immune-modulating agent.
• the present invention provides a use of an LSDl inhibitor for enhancing the efficacy of an anti-infective agent.
• a composition is provided comprising an LSDl inhibitor and an anti-infective agent.
• a method of enhancing an immune response in a subject to a target antigen by an immune-modulating agent comprising administering an LSDl inhibitor to the subject.
• a method of inhibiting PD-L1 and/or PD-L2 activity comprising contacting a PD-L1 and/or PD-L2 overexpressing cell with an LSDl inhibitor.
• an LSDl inhibitor for inhibiting PD-L1 and/or PD-L2 activity comprising contacting a PD-L1 and/or PD-L2 overexpressing cell with an LSD1 inhibitor.
• a method of inhibiting the nuclear translocation of PD-Ll and/or PD-L2 in a PD-Ll and/or PD-L2 overexpressing cell comprising contacting the PD-Ll and/or PD-L2 overexpressing cell with an LSD1 inhibitor.
• the present inventors have also conceived that proteinaceous molecules based on a subsequence of the LSD1 polypeptide and structurally-related molecules inhibit LSD1 activity, including the translocation of LSD1 into the nucleus of a cell, and are also useful therefore for inhibiting PD-Ll and/or PD-L2 activity, as broadly described above and elsewhere herein.
• proteinaceous molecules are also proposed to inhibit the phosphorylating activity of a PKC, especially PKC- ⁇ .
• PKC phosphorylating activity of a PKC
• These molecules have also been shown to have significant activity in inhibiting EMT, in inhibiting formation and maintenance of cancer stem cell and non- cancer stem cell tumor cells, and in inducing MET, which makes them useful in treating a range of conditions, associated with LSD1 overexpression, such as cancer.
• Another aspect of the invention provides a method of inhibiting the
• PKC protein kinase C
• a method of altering at least one of (i) formation; (ii) proliferation ; (iii) maintenance; (iv) EMT; or (v) M ET of a PKC overexpressing cell comprising contacting said PKC overexpressing cell with a formation-, proliferation-, maintenance-, EMT- or M ET-modulating amount of an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence
• the present invention also provides a method of treating or preventing a cancer in a subject, wherein the cancer comprises at least one PKC overexpressing cell, comprising administering to the subject an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence corresponding to residues 108 to 118 of LSD1.
• a method of inhibiting PKC phosphorylation of LSD1 in an LSD1 overexpressing cell comprising contacting the LSD1 overexpressing cell with an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence corresponding to residues 108 to 118 of LSD1.
• a method of inhibiting an activity of LSD1 comprising contacting an LSD1 overexpressing cell with an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence
• a further aspect of the invention provides a method of inhibiting the nuclear translocation of LSD1 in an LSD1 overexpressing cell, comprising contacting the LSD1
• overexpressing cell with an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence corresponding to residues 108 to 118 of LSD1.
• a method of altering at least one of (i) formation; (ii) proliferation ; (iii) maintenance; (iv) EMT; or (v) MET of an LSD1 overexpressing cell comprising contacting said LSD1 overexpressing cell with a formation-, proliferation-, maintenance-, EMT- or M ET-modulating amount of an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence
• the invention also extends to a method of treating or preventing a cancer in a subject, wherein the cancer comprises at least one LSDl overexpressing cell, comprising administering to the subject an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence corresponding to residues 108 to 118 of LSDl.
• the present invention in another aspect, provides a use of an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence
• the present invention also contemplates a use of an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence corresponding to residues 108 to 118 of LSDl for treating or preventing a cancer in a subject, wherein the cancer comprises at least one PKC overexpressing cell.
• the present invention provides a use of an isolated or purified proteinaceous molecule comprising a sequence corresponding to residues 108 to 118 of LSDl in the manufacture of a medicament for treating or preventing a cancer in a subject, wherein the cancer comprises at least one PKC overexpressing cell.
• the present invention provides a use of an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence
• the present invention contemplates a use of an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence corresponding to residues 108 to 118 of LSDl in the manufacture of a medicament for inhibiting an activity of LSDl.
• the present invention also extends to a use of an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence corresponding to residues 108 to 118 of LSDl for altering at least one of (i) formation ; (ii) proliferation ; (iii) maintenance; (iv) EMT; or (v) M ET of an LSDl overexpressing cell.
• the cancer comprises at least one LSDl overexpressing cell.
• Yet a further aspect of the invention provides a use of an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence
• Another aspect of the invention provides an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence corresponding to residues 108 to 118 of LSDl for use in inhibiting the phosphorylating activity of a PKC in a PKC overexpressing cell.
• an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence
• an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence corresponding to residues 108 to 118 of LSD1 for use in treating or preventing a cancer in a subject, wherein the cancer comprises at least one PKC overexpressing cell.
• the invention provides an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence corresponding to residues 108 to 118 of LSD1 for use in inhibiting PKC phosphorylation of LSD1 in an LSD1 overexpressing cell.
• an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence
• the invention also contemplates an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence corresponding to residues 108 to 118 of LSD1 for use in inhibiting the nuclear translocation of LSD1 in an LSD1 overexpressing cell.
• proteinaceous molecule comprising, consisting or consisting essentially of a sequence
• the invention provides an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence corresponding to residues 108 to 118 of LSD1 for use in treating or preventing a cancer in a subject, wherein the cancer comprises at least one LSD1 overexpressing cell.
• Zi and Z 2 are independently absent or are independently selected from at least one of a proteinaceous moiety comprising from about 1 to about 50 amino acid residues (and all integer residues in between), and a protecting moiety;
• Xi is selected from small amino acid residues, including S, T, A, G and modified forms thereof; wherein the proteinaceous molecule is other than a proteinaceous molecule consisting of the amino acid sequence of SEQ ID NO : 4:
• Figure 1 is a photographic and graphical representation of expression of PD-L1 and LSD1 phosphorylated at serine 111 (LSDlsll lp) in MCF7 [stimulated with PMA (MCF7ST) and non- stimulated (MCF7NS)] and MDA-MB-231 (MDA) breast cancer cell lines.
• Total nuclear fluorescence (TNFI), total cytoplasmic fluorescence (TCFI), the nuclear bias (Fn/c) and the degree of co- localization (PCC) is presented.
• Figure 2 is a photographic and graphical representation of expression of LSD1 phosphorylated at serine 111 (LSDlp) and PD-L2 in M DA-M B-231 breast cancer cell lines.
• Total nuclear fluorescence (TNFI), total cytoplasmic fluorescence (TCFI), the nuclear bias (Fn/c) and the degree of co-localization (PCC) is presented.
• Figure 3 depicts the expression of CD44, PD-L1 and PD-L2 in MCF7 [adherent cells stimulated with PMA (MCF7 ST AD) ; suspension cells stimulated with PMA (MCF7 ST SUS) ; and non-stimulated (MCF7 NS)] and M DA-M B-231 cells. FACS plots of inducible MCF7 cells non- stimulated, stimulated with PMA or stimulated with PMA and TGF- ⁇ and stained for PD-L1 are presented in Figure 3A.
• Figure 3B depicts the expression of CD44, PD-L1 and PD-L2 mRNA in MCF7 and MDA-MB-231 cells.
• Figure 3C depicts the expression of CD44, PD-L1 and PD-L2 mRNA in MCF7 cells treated with LSD1 siRNA.
• Figure 4 is a graphical and photographic representation of the effect of peptide inhibitors LI, L2 and L3 on the expression and nuclear localization of LSD1 phosphorylated at serine 111 (LSDlp) in stimulated (MCF7ST) and non-stimulated (MCF7NS) MCF7 cells and MDA-MB-231 cells (M DA). Total nuclear fluorescence (TNFI) was determined.
• Figure 5 is a graphical and photographic representation of the effect of peptide inhibitors LI, L2 and L3 on the expression and nuclear localization of PD-L1 in stimulated (MCF7ST) and non- stimulated (MCF7NS) MCF7 cells.
• Total nuclear fluorescence (TN FI), total cytoplasmic fluorescence (TCFI) and the nuclear bias (Fn/c) were determined.
• Figure 6 is a graphical and photographic representation of the effect of peptide inhibitors LI, L2 and L3 on the expression and nuclear localization of PD-L1 in M DA-M B-231 cells.
• Total nuclear fluorescence (TNFI), total cytoplasmic fluorescence (TCFI) and the nuclear bias (Fn/c) were determined.
• Figure 7 is a graphical and photographic representation of the effect of treatment of a mouse M DA-MB-231 xenograft with vehicle (Con), Abraxane (60 mg/kg, 30 mg/kg and 10 mg/kg) (Abrax) or Docetaxel (10 mg/kg and 4 mg/kg) (Doc).
• Figure 7A shows the volume of the tumors over time during treatment.
• Figure 7B shows the nuclear fluorescent signal (TNFI) of (i) LSD1 phosphorylated at serine 111 (LSDlsll lp), (ii) epidermal growth factor receptor (EGFR), and (iii) SNAIL in Abraxane or Docetaxel treated cells relative to the vehicle alone.
• TNFI nuclear fluorescent signal
• Figure 8 is a graphical and photographic representation of the effect of treatment with vehicle (Con), Abraxane (60 mg/kg) (Abrax), Phenelzine (41 mg/kg, 27 mg/kg or 13.5 mg/kg) (Phenel) or a combination thereof on mouse xenograft MDA-M B-231 cells.
• Figures 8A-E depict the tumor size and volume over time.
• Figure 9 is a graphical and photographic representation of the effect of treatment with Abraxane (60 mg/kg) (Abrax), Phenelzine (41 mg/kg) (Phenel) or a combination thereof on mouse xenograft M DA-MB-231 cells.
• Figure 9 depicts the expression of LSD1 phosphorylated at serine 111 (LSDls 111p), cytokeratin and PD-L1. This figure assesses the total nuclear fluorescence
• TNFI total cytoplasmic fluorescence
• TCFI total cytoplasmic fluorescence
• Fn/c nuclear bias
• PCC degree of co-localization
• Figure 10 is a graphical and photographic representation of the effect of treatment with Abraxane (60 mg/kg) (Abrax), Phenelzine (41 mg/kg) (Phenel) or a combination thereof on mouse xenograft M DA-MB-231 cells. This figure assess the nuclear (TNFI) and cytoplasmic (TCFI) expression of EGFR and cell surface vimentin (CSV).
• Figure 11 is a graphical and photographic representation of the effect of treatment with vehicle (Mock; Group A), Abraxane (60 mg/kg) (Group B), or Docetaxel (10 mg/kg) (Group E) on mouse xenograft M DA-MB-231 cells.
• This figure presents the expression of PD-L2 and the mesenchymal marker, M ET.
• This figure assesses the total nuclear fluorescence (TNFI), total cytoplasmic fluorescence (TCFI), nuclear bias (Fn/c) and the degree of co-localization (PCC).
• TNFI total nuclear fluorescence
• TCFI total cytoplasmic fluorescence
• Fn/c nuclear bias
• PCC degree of co-localization
• Figure 12 presents representative images of circulating tumor cells isolated from patient liquid biopsies.
• the images show the expression of SNAIL, a transcription factor implicated in aggressive cancer with a mesenchymal state; vimentin and cyctokeratin, which are markers for circulating tumor cells; LSD1 phosphorylated at serine 111 (LSDls111p) ; PD-L1 ; and PD-L2.
• Figure 13 is a photographic ( Figure 13A) and graphical ( Figure 13B) representation of the expression of LSD1 phosphorylated at serine 111 (LSDls111p), PD-L1 and cell surface vimentin (CSV) in circulating tumor cells isolated from breast cancer patient liquid biopsies.
• Total nuclear fluorescence (TNFI), cytoplasmic fluorescence (TCFI), nuclear bias (Fn/c) and the degree of co-localization (PCC) are depicted.
• Figure 14 is a photographic and graphical representation of the expression of of LSD1 phosphorylated at serine 111 (LSDlsll lp), PD-L2 and cytokeratin in circulating tumor cells isolated from breast cancer patient liquid biopsies.
• Total nuclear fluorescence (TNFI), cytoplasmic fluorescence (TCFI), nuclear bias (Fn/c) and the degree of co-localization (PCC) are depicted.
• Figure 15 is a photographic ( Figure 15A) and graphical ( Figure 15B) representation of the expression of cell surface vimentin, SNAIL and PD-L1 in circulating tumor cells isolated from breast cancer patient liquid biopsies. Total nuclear fluorescence (TNFI), cytoplasmic fluorescence (TCFI), nuclear bias (Fn/c) and the degree of co-localization (PCC) are depicted.
• TNFI Total nuclear fluorescence
• TCFI cytoplasmic fluorescence
• Fn/c nuclear bias
• PCC degree of co-localization
• Figure 16 depicts the total cytoplasmic expression (TCFI) of LSD1 phosphorylated at serine 111 (LSDls111p), cytokeratin and PD-L2 in circulating tumor cells isolated from breast cancer patient liquid biopsies in response to treatment with Pargyline (Parg) and Phenelzine.
• TCFI total cytoplasmic expression
• Figure 17 is a photographic and graphical representation of the expression of cell surface vimentin, PD-L1 and SNAIL in circulating tumor cells isolated from breast cancer patient liquid biopsies in response to treatment with Pargyline (Figure 17A) and Phenelzine (Figure 17B) in comparison to vehicle (Mock).
• the total cell fluorescence (TCFI) is presented.
• Figure 18 presents the effect of cyclin-dependent kinase (CDK) inhibitors Palbociclib
• Figure 19 is a graphical and photographic representation of the effect of three peptide LSD1 inhibitors, LI, L2 and L3, on nuclear translocation of LSD1 in M DA-MB-231 cells in comparison to control (Ctrl).
• the nuclear bias (Fn/c) of LSD1 expression is presented.
• Figure 20 depicts the effect of the LSD1 peptide inhibitors, LI, L2 and L3 (50 ⁇ and 100 ⁇ ) on cancer stem cell (CSC) formation in MCF7 cells. FACS data and graphical
• Figure 21 depicts the effect of the LSDl peptide inhibitors, LI, L2 and L3 (50 ⁇ and 100 ⁇ ) on cancer stem cell (CSC) formation in M DA-MB-231 cells. FACS data and graphical representations thereof are presented.
• Figure 22 is a photographic and graphical representation of the effect of the LSDl peptide inhibitors, LI, L2 and L3 (50 ⁇ and 100 ⁇ ) on the expression of PD-L1 in MDA-M B-231 (M DA) cells in comparison to control (Mock). Total cell fluorescence (TFI) is presented.
• Figure 23 is a photographic and graphical representation of the effect of the LSDl peptide inhibitors, LI, L2 and L3 on the expression of epidermal growth factor receptor (EGFR) in M DA-MB-231 (MDA) cells in comparison to control (Pep-Ctrl). Total nuclear fluorescence (TNFI) is presented.
• EGFR epidermal growth factor receptor
• MDA-MB-231 MDA-MB-231
• Figure 24 is a photographic and graphical representation of the effect of LI, L2 and L3 on the expression of M ET (a mesenchymal marker) in MDA-M B-231 (M DA) cells in comparison to control (Pep-Ctrl). Total nuclear fluorescence (TNFI) is presented.
• M ET a mesenchymal marker
• Figure 25 is a photographic and graphical representation of the expression of PKC- ⁇ and LSDl in non-stimulated (MCF7NS), PMA stimulated adherent (MCF7 ST) and PMA stimulated floating (MCF7 FLT) MCF7 cells, and MDA-MB-231 (MDA) cells. Data represent the mean ⁇ SEM .
• the average fluorescence signal intensity for the indicated pair of antibodies was plotted for each point on the line with standard error.
• Signal was plotted to compare how the signals for each antibody varied compared to the opposite antibody.
• the degree of co-localization (PCC) was determined for each plot profile.
• TNFI Total nuclear fluorescence
• Figure 26 is a photographic and graphical representation of the expression of LSDl phosphorylated at serine 111 (LSDlsll lp) in non-stimulated (MCF7NS) and stimulated (MCF7ST) MCF7 cells and MDA-M B-231 (M DA) cells in response to treatment with the PKC- ⁇ inhibitors C27 or BIM.
• Total nuclear fluorescence (TNFI) is presented. Data shown represented mean ⁇ SEM for at least 20 individual cells.
• Figure 27 depicts the effect of Abraxane (60 mg/kg) and Docetaxel (10 mg/kg) treatment on expression of LSDl phosphorylated at serine 111 (LSDls111p) in xenografted M DA- M B-231 cells in comparison to control (Mock). Representative images and a graph presenting the total nuclear fluorescence (TNFI) are presented. Data shown represents the mean ⁇ SEM for at least 20 individual cells.
• Figure 28 is a photographic and graphical representation of the effect of LSDl siRNA (si- LSD1) on the expression of LSDl and SNAIL in PMA stimulated (ST) and non-stimulated (NS) MCF7 cells in comparison to control (Mock). Data presented represents the mean ⁇ SEM for at least 20 cells.
• si- LSD1 LSDl siRNA
• Figure 29 is a photographic and graphical representation of the effect of inhibitors of the catalytic activity of LSDl, NCD36 (NCB) and pargyline (Parg), on the expression of LSDl and SNAIL in PMA stimulated (ST) and non-stimulated (NS) MCF7 cells in comparison to control (Mock). Data presented represents the mean ⁇ SEM for at least 20 cells.
• Figure 30 is a photographic and graphical representation of the effect of LSDl siRNA (si- LSD1), pargyline (Parg) and NCD36 (NCB) on the expression of LSDl and SNAIL in M DA-M B-231 cells in comparison to control (Mock). Data presented represents the mean ⁇ SEM for at least 20 cells.
• Figure 31 is a photographic and graphical representation of the effect of the peptide inhibitors, LI, L2 and L3, on the expression of LSDl in M DA-MB-231 cells in comparison to control (Ctrl). Data presented represents the mean ⁇ SEM for at least 20 cells. The nuclear bias (Fn/c) of LSDl expression is presented.
• “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
• administering concurrently or “administered concurrently” and the like refer to the administration of a single composition containing two or more actives, or the administration of each active as separate compositions and/or delivered by separate routes either contemporaneously or simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such actives are administered as a single composition.
• simultaneous is meant that the active agents are administered at substantially the same time, and desirably together in the same formulation.
• temporary it is meant that the active agents are administered closely in time, e.g. , one agent is administered within from about one minute to within about one day before or after another.
• any contemporaneous time is useful. However, it will often be the case that when not administered simultaneously, the agents will be administered within about one minute to within about eight hours and preferably within less than about one to about four hours. When administered contemporaneously, the agents are suitably administered at the same site on the subject.
• the term "same site” includes the exact location, but can be within about 0.5 to about 15 centimeters, preferably from within about 0.5 to about 5 centimeters.
• the term "separately" as used herein means that the agents are administered at an interval, for example, at an interval of about one day to several weeks or months.
• the active agents may be administered in any order.
• agent or “modulatory agent” includes a compound that induces a desired pharmacological and/or physiological effect.
• the term also encompass pharmaceutically acceptable and pharmacologically active ingredients of those compounds specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like.
• agent includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc.
• agent is not to be construed narrowly but extends to small molecules, proteinaceous molecules such as peptides, polypeptides and proteins as well as compositions comprising them and genetic molecules such as RNA, DNA and mimetics and chemical analogs thereof as well as cellular agents.
• agent includes a cell that is capable of producing and secreting a polypeptide referred to herein as well as a polynucleotide comprising a nucleotide sequence that encodes that polypeptide.
• the term “agent” extends to nucleic acid constructs including vectors such as viral or non-viral vectors, expression vectors and plasmids for expression in and secretion in a range of cells.
• alkyl refers to a straight chain, branched or cyclic saturated hydrocarbon group having 1 to 10 carbon atoms. Where appropriate, the alkyl group may have a specified number of carbon atoms, for example, C 1- C 6 alkyl which includes alkyl groups having 1, 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement.
• alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, / ' -propyl, n-butyl, / ' -butyl, t-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 4-methylbutyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4- methylpentyl, 5-methylpentyl, 2-ethylbutyl, 3-ethylbutyl, heptyl, octyl, nonyl, decyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
• alkenyl refers to a straight-chain, branched or cyclic hydrocarbon group having one or more double bonds between carbon atoms and having 2 to 10 carbon atoms. Where appropriate, the alkenyl group may have a specified number of carbon atoms. For example, C 2 -C 6 as in "C 2 -C 6 alkenyl” includes groups having 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement.
• alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, butadienyl, pentenyl, pentadienyl, hexenyl, hexadienyl, heptenyl, octenyl, nonenyl, decenyl, cyclopentenyl, cyclohexenyl and cyclohexadienyl.
• alkyl means alkyl as defined above which is substituted with an aryl group as defined above, e.g.,-CH2phenyl,-(CH2)2Phenyl,-(CH 2 )3phenyl,
• aromatic or aryl is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 atoms in each ring, wherein at least one ring is aromatic.
• aryl elements include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl.
• substituents may be defined with a range of carbons that includes zero, such as (C 0 -C 6 )alkylene-aryl. If aryl is taken to be phenyl, this definition would include phenyl itself as well as, for example, -CH 2 Ph,-CH 2 CH 2 Ph, CH(CH 3 )CH 2 CH(CH 3 )Ph.
• the compounds described herein may possess asymmetric centers and are therefore capable of existing in more than one stereoisomeric form.
• the invention thus also relates to compounds in substantially pure isomeric form at one or more asymmetric centers e.g. greater than about 90% ee, such as about 95% or 97% ee or greater than 99% ee, as well as mixtures, including racemic mixtures, thereof.
• Such isomers may be naturally occurring or may be prepared by asymmetric synthesis, for example using chiral intermediates, or by chiral resolution.
• antigen as used herein to refer to all, or part of, a protein, peptide, or other molecule or macromolecule capable of eliciting an immune response in a vertebrate animal, especially a mammal. Such antigens are also reactive with antibodies from animals immunized with that protein, peptide, or other molecule or macromolecule.
• antigen-binding molecule is used herein to refer to a molecule that has binding affinity for a target antigen. It will be understood that this term extends to
• immunoglobulins immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity.
• cancer stem cell refers to a cell that has tumor-initiating and tumor- sustaining capacity, including the ability to extensively proliferate, form new tumors and maintain cancer development, i.e. cells with indefinite proliferative potential that drive the formation and growth of tumors. CSCs are biologically distinct from the bulk tumor cells and possess
• cancer stem cell includes both gene alteration in stem cells (SCs) and gene alteration in a cell which becomes a CSC.
• SCs stem cells
• the CSCs are breast CSCs, which are suitably CD24 + CD44 + , illustrative examples of which include CD44 high CD24 l0W .
• catalytic activity in relation to LSD1 is used herein to refer to the demethylation of a methylated lysine residue in a protein, such as a histone, especially histone 3.
• the term “consisting essentially of” in the context of a specific amino acid sequence disclosed herein, includes within its scope about 1 to about 50 optional amino acids (and all integer optional amino acids in between) upstream of the specific amino acid sequence and/or about 1 to about 50 optional amino acids (and all integer optional amino acids in between) downstream of the specific amino acid sequence.
• a sequence such as a nucleic acid or amino acid sequence, that displays substantial sequence identity to a reference sequence (e.g. at least about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence identity to all or a portion of the reference nucleic acid sequence) or an amino acid sequence that displays substantial sequence similarity or identity to a reference amino acid sequence (e.g.
• derivative is meant a molecule, such as a polypeptide, that has been derived from the basic molecule by modification, for example by conjugation or complexing with other chemical moieties or by post-translational modification techniques as would be understood in the art.
• derivative also includes within its scope alterations that have been made to a parent sequence including additions or deletions that provide for functionally equivalent molecules.
• dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable vehicle.
• an effective amount in the context of treating or preventing a condition is meant the administration of an amount of an agent or composition to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and/or treating existing symptoms, of that condition.
• the effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
• effector T cell includes T cells which function to eliminate antigen (e.g. by producing cytokines which modulate the activation of other cells or by cytotoxic activity).
• Immunoenhancement means to increase the animal's capacity to respond to foreign or disease-specific antigens (e.g. cancer antigens) i.e. those cells primed to attack such antigens are increased in number, activity, and ability to detect and destroy the those antigens.
• Antigens e.g. cancer antigens
• Strength of immune response is measured by standard tests including : direct measurement of peripheral blood lymphocytes by means known to the art; natural killer cell cytotoxicity assays (see, e.g., Provinciali et al. (1992) J. Immunol.
• any statistically significant increase in strength of immune response as measured by the foregoing tests is considered “enhanced immune response” or “immunoenhancement” as used herein.
• Enhanced immune response is also indicated by physical manifestations such as fever and inflammation, as well as healing of systemic and local infections, and reduction of symptoms in disease, i.e., decrease in tumor size, alleviation of symptoms of a disease or condition including, but not restricted to, leprosy, tuberculosis, malaria, naphthous ulcers, herpetic and papillomatous warts, gingivitis, artherosclerosis, the concomitants of AIDS such as Kaposi's sarcoma, bronchial infections, and the like.
• Such physical manifestations also define “enhanced immune response” or “immunoenhancement” as used herein.
• EMT epithelial-to-mesenchymal transition
• Classes of molecules that change in expression, distribution and/or function during EMT, and that are causally involved, include growth factors (e.g. transforming growth factor (TGF)-p, wnts), transcription factors (e.g. SNAI, SMAD, LEF and nuclear ⁇ -catenin), molecules of the cell-to-cell adhesion axis (cadherins, catenins), cytoskeletal modulators (Rho family) and extracellular proteases (matrix metalloproteinases, plasminogen activators) (refer to Thompson and Newgreen, Cancer Res. 2005; 65(14) : 5991-5).
• growth factors e.g. transforming growth factor (TGF)-p, wnts
• transcription factors e.g. SNAI, SMAD, LEF and nuclear ⁇ -catenin
• cadherins, catenins e.g. cytoskeletal modulators (Rho family) and extracellular proteases (matrix metallo
• epithelium refers to the covering of internal and external surfaces of the body, including the lining of vessels and other small cavities. It consists of a collection of epithelial cells forming a relatively thin sheet or layer due to the constituent cells being mutually and extensively adherent laterally by cell-to-cell junctions. The layer is polarized and has apical and basal sides. Despite the tight regimentation of the epithelial cells, the epithelium does have some plasticity and cells in an epithelial layer can alter shape, such as change from flat to columnar or pinch in at one end and expand at the other. However, these tend to occur in cell groups rather than individually (refer to Thompson and Newgreen, Cancer Res. 2005;
• expression refers the biosynthesis of a gene product.
• expression involves transcription of the coding sequence into mRNA and translation of mRNA into one or more polypeptides.
• expression of a non-coding sequence involves transcription of the non-coding sequence into a transcript only.
• expression is also used herein to refer to the presence of a protein or molecule in a particular location and, thus, may be used interchangeably with “localization”.
• expression vector any genetic element capable of directing the transcription of a polynucleotide contained within the vector and suitably the synthesis of a peptide or polypeptide encoded by the polynucleotide. Such expression vectors are known to practitioners in the art.
• group as applied to chemical species refers to a set of atoms that forms a portion of a molecule. In some instances, a group can include two or more atoms that are bonded to one another to form a portion of a molecule. A group can be monovalent or polyvalent (e.g. bivalent) to allow bonding to one or more additional groups of a molecule.
• a monovalent group can be envisioned as a molecule with one of its hydrogen atoms removed to allow bonding to another group of a molecule.
• a group can be positively or negatively charged.
• a positively charged group can be envisioned as a neutral group with one or more protons (i.e. H + ) added, and a negatively charged group can be envisioned as a neutral group with one or more protons removed.
• Non-limiting examples of groups include, but are not limited to, alkyl groups, alkylene groups, alkenyl groups, alkenylene groups, alkynyl groups, alkynylene groups, aryl groups, arylene groups, iminyl groups, iminylene groups, hydride groups, halo groups, hydroxy groups, alkoxy groups, carboxy groups, thio groups, alkylthio groups, disulfide groups, cyano groups, nitro groups, amino groups, alkylamino groups, dialkylamino groups, silyl groups, and siloxy groups.
• Groups such as alkyl, alkenyl, alkynyl, aryl, and heterocyclyl, whether used alone or in a compound word or in the definition of a group may be optionally substituted by one or more substituents.
• optional substituents include alkyl, preferably C 1-8 alkyl (e.g. C 1-6 alkyl such as methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl), hydroxy C 1-8 alkyl (e.g. hydroxymethyl, hydroxyethyl, hydroxypropyl), alkoxyalkyl (e.g.
• C 1-8 alkyl e.g. C 1-6 alkyl such as methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl
• hydroxy C 1-8 alkyl e.g. hydroxymethyl, hydroxyethyl, hydroxypropyl
• alkoxyalkyl e.g.
• C 1-6 alkyl such as methylamino, ethylamino, propylamino
• diC 1 _ 8 alkylamino e.g. C 1-6 alkyl, such as dimethylamino, diethylamino, dipropylamino
• acylamino e.g. NHC(0)CH 3
• phenylamino wherein phenyl itself may be further substituted as described herein
• nitro, formyl, -C(0)-C 1-8 alkyl e.g. C 1- 6 alkyl, such as acetyl
• 0-C(0)-alkyl e.g.
• C 1-6 alkyl such as acetyloxy
• benzoyl wherein the CH 2 and/or phenyl group itself may be further substituted
• C 1-6 alkyl such as methyl ester, ethyl ester, propyl ester, butyl ester
• C0 2 phenyl wherein phenyl itself may be further substituted
• CONH 2 CONHphenyl (wherein phenyl itself may be further substituted as described herein)
• CONHbenzyl wherein the CH 2 and/or phenyl group may be further substituted as described herein
• CONH C 1-8 alkyl e.g. C 1-6 alkyl such as methyl amide, ethyl amide, propyl amide, butyl amide
• CONH C 1-8 alkylamine e.g.
• C 1-6 alkyl such as aminomethyl amide, aminoethyl amide, aminopropyl amide, aminobutyl amide), -C(0)heterocyclyl (e.g. -C(0)-l-piperidine, -C(0)-l-piperazine, -C(0)-4-morpholine), -C(0)heteroaryl (e.g. -C(0)-1- pyridine, -C(0)-l-pyridazine, -C(0)-l-pyrimidine, -C(O)-l-pyrazine), CONHdiC 1-8 alkyl (e.g. C 1-6 alkyl).
• -C(0)heterocyclyl e.g. -C(0)-l-piperidine, -C(0)-l-piperazine, -C(0)-4-morpholine
• -C(0)heteroaryl e.g. -C(0)-1- pyridine, -C(0)-l-pyridazine
• Heteroaralkyl means alkyl as defined above which is substituted with a heteroaryl group, e.g.,-CH 2 pyridinyl,-(CH 2 ) 2 Pyrimidinyl,-(CH 2 ) 3 imidazolyl, and the like, and derivatives thereof.
• heteroaryl or “heteroaromatic”, as used herein, represents a stable monocyclic or bicyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of 0, N and S.
• Heteroaryl groups within the scope of this definition include but are not limited to : acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, bezofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline.
• heteroaryl is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl.
• heterocyclyl and “heteroaryl” include, but are not limited to, the following : benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazoyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoqu inolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyr
• heteroarylene refers to a bivalent monocyclic or multicyclic ring system, preferably of about 3 to about 15 members where one or more, more preferably 1 to 3 of the atoms in the ring system is a heteroatom, that is, an element other than carbon, for example, nitrogen, oxygen and sulfur atoms.
• the heteroarylene group may be optionally substituted with one or more, preferably 1 to 3, aryl group substituents.
• Exemplary heteroarylene groups include, for example, 1,4-imidazolylene.
• heterocycle means a 5-to 10-membered nonaromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of 0, N and S, and includes bicyclic groups.
• heterocyclylalkyl means alkyl as defined above which is substituted with a heterocycle group, e.g.,-CH 2 pyrrolidin-l-yl,-(CH 2 )2Piperidin-l-yl, and the like, and derivatives thereof.
• the term “high”, as used herein, refers to a measure that is greater than normal, greater than a standard such as a predetermined measure or a subgroup measure or that is relatively greater than another subgroup measure.
• CD44 high refers to a measure of CD44 that is greater than a normal CD44 measure. Consequently, "CD44 high " always corresponds to, at the least, detectable CD44 in a relevant part of a subject's body or a relevant sample from a subject's body.
• a normal measure may be determined according to any method available to one skilled in the art.
• the term “high” may also refer to a measure that is equal to or greater than a predetermined measure, such as a predetermined cutoff. If a subject is not “high” for a particular marker, it is “low” for that marker. In general, the cut-off used for determining whether a subject is "high” or “low” should be selected such that the division becomes clinically relevant.
• the term "hormone receptor negative (HR-) tumor” means a tumor that does not express a receptor for a hormone that stimulates the proliferation, survival or viability of the tumor above a certain threshold as determined by standard methods (e.g. immunohistochemical staining of nuclei in the patients biological samples).
• the threshold may be measured, for example, using an Allred score or gene expression. See, e.g. Harvey et al. (1999, J Clin Oncol, 17 : 1474-1481) and Badve et al. (2008, J Clin Oncol, 26(15) : 2473-2481).
• the tumor does not express an estrogen receptor (ER-) and/or a progesterone receptor (PR-).
• the term "hormone receptor positive (HR+) tumor” means a tumor that expresses a receptor for a hormone that stimulates the proliferation, survival or viability of the tumor above a certain threshold as determined by standard methods (e.g. immunohistochemical staining of nuclei in the patients biological samples).
• the threshold may be measured, for example, using an Allred score or gene expression. See, e.g., Harvey et al. (1999, J Clin Oncol, 17 : 1474-1481) and Badve et al. (2008, J Clin Oncol, 26(15) : 2473-2481).
• the tumor expresses an estrogen receptor (ER) and/or a progesterone receptor (PR).
• ER estrogen receptor
• PR progesterone receptor
• host cell includes an individual cell or cell culture which can be or has been a recipient of any recombinant vector(s) or isolated polynucleotide of the invention.
• Host cells include progeny of a single host cell and the progeny may not necessarily be completely identical
• a host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention.
• a host cell which comprises a recombinant vector of the invention is a recombinant host cell.
• Hybridization is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid.
• Complementary base sequences are those sequences that are related by the base-pairing rules. In DNA, A pairs with T and C pairs with G. In RNA U pairs with A and C pairs with G. In this regard, the terms "match" and
• mismatch refers to the hybridization potential of paired nucleotides in
• the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds.
• hydrogen bonding which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds.
• nucleobases complementary nucleoside or nucleotide bases
• adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
• Hybridization can occur under varying
• hydrocarbyl as used herein includes any radical containing carbon and hydrogen including saturated, unsaturated, aromatic, straight or branched chain or cyclic including polycyclic groups. Hydrocarbyl includes but is not limited to C 1- C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 - C 10 cycloalkyl, aryl such as phenyl and naphthyl, Ar(d-C 8 )alkyl such as benzyl, any of which may be optionally substituted.
• immuno-interactive includes reference to any interaction, reaction, or other form of association between molecules and in particular where one of the molecules is, or mimics, a component of the immune system.
• inhibitor refers to an agent that decreases or inhibits at least one function or biological activity of a target molecule.
• an LSD1 inhibitor may decrease or reduce at least one function or biological activity of LSD1.
• LSD1 inhibitors may inhibit or reduce the nuclear translocation of LSD1, may inhibit or reduce the catalytic activity of LSD1, may inhibit or reduce the phosphorylation of LSD1, and/or may inhibit or reduce the expression of LSD1.
• the term “LSD1 inhibitor” refers to an agent that inhibits the nuclear translocation of LSD1.
• isolated refers to material that is substantially or essentially free from components that normally accompany it in its native state.
• isolated proteinaceous molecule refers to in vitro isolation and/or purification of a proteinaceous molecule from its natural cellular environment and from association with other components of the cell.
• Substantially free means that a preparation of proteinaceous molecule is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% pure.
• the preparation of proteinaceous molecule has less than about 30, 25, 20, 15, 10, 9, 8, 7 , 6, 5, 4, 3, 2 or 1% (by dry weight), of molecules that are not the subject of this invention (also referred to herein as "contaminating molecules").
• the proteinaceous molecule When the proteinaceous molecule is recombinantly produced, it is also desirably substantially free of culture medium, i.e., culture medium represents less than about 20, 15, 10, 5, 4, 3, 2 or 1% of the volume of the preparation.
• culture medium represents less than about 20, 15, 10, 5, 4, 3, 2 or 1% of the volume of the preparation.
• the invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.
• lower alkyl refers to straight and branched chain alkyl groups having from 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, / ' -propyl, n-butyl, t-butyl, sec-butyl, n-pentyl, n-hexyl, 2-methylpentyl, and the like.
• the lower alkyl group is methyl or ethyl.
• lower alkoxy refers to straight and branched chain alkoxy groups having from 1 to 6 carbon atoms, such as methoxy, ethoxy, n-propoxy, / ' -propoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 2-methyl-pentoxy, and the like.
• the lower alkoxy group is methoxy or ethoxy.
• LSD1 overexpressing cell refers to a vertebrate cell, particularly a mammalian or avian cell, especially a mammalian cell, that expresses LSD1 at a detectably greater level than a normal cell.
• the cell may be a vertebrate cell, such as a primate cell; an avian cell; a livestock animal cell such as a sheep cell, cow cell, horse cell, deer cell, donkey cell and pig cell; a laboratory test animal cell such as a rabbit cell, mouse cell, rat cell, guinea pig cell and hamster cell; a companion animal cell such as a cat cell and dog cell; and a captive wild animal cell such as a fox cell, deer cell and dingo cell.
• the LSD1 overexpressing cell is a human cell.
• the LSD1 overexpressing cell is a cancer stem cell or a non-cancer stem cell tumor cell; preferably a cancer stem cell tumor cell. Overexpression can also be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a normal cell or comparison cell (e.g. a breast cell).
• M ET mesenchymal-to-epithelial transition
• M ET mesenchymal cells to planar arrays of polarized cells called epithelia.
• M ET is the reverse process of EMT. METs occur in normal development, cancer metastasis and induced pluripotent stem cell reprogramming.
• meenchyme refers to the part of the embryonic mesoderm, consisting of loosely packed, unspecialized cells set in a gelatinous ground substance, from which connective tissue, bone, cartilage and the circulatory and lymphatic systems develop.
• Mesenchyme is a collection of cells which form a relatively diffuse tissue network. Mesenchyme is not a complete cellular layer and the cells typically have only points on their surface engaged in adhesion to their neighbors. These adhesions may also involve cadherin association (see
• modulating is meant increasing or decreasing, either directly or indirectly, the level or functional activity of a target molecule.
• an agent may indirectly modulate the level/activity by interacting with a molecule other than the target molecule.
• indirect modulation of a gene encoding a target polypeptide includes within its scope modulation of the expression of a first nucleic acid molecule, wherein an expression product of the first nucleic acid molecule modulates the expression of a nucleic acid molecule encoding the target polypeptide.
• overexpressed interchangeably refer to a gene (e.g. LSD1 gene or PKC gene) that is transcribed or translated at a detectably greater level, usually in a cancer cell, in comparison to a normal cell.
• Overexpression therefore, refers to both overexpression of protein and RNA (due to increased transcription, post transcriptional processing, translation, post translational processing, altered stability and altered protein degradation), as well as local overexpression due to altered protein traffic patterns (increased nuclear localization) and augmented functional activity, for example, as in an increased enzyme hydrolysis of substrate.
• Overexpression can also be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a normal cell or comparison cell (e.g. a breast cell).
• operably linked means placing a structural gene under the regulatory control of a regulatory element including, but not limited to, a promoter, which then controls the transcription and optionally translation of the gene.
• a regulatory element including, but not limited to, a promoter
• the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e. the genes from which it is derived.
• pharmaceutically acceptable carrier a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction.
• Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, transfection agents and the like.
• a "pharmacologically acceptable" salt, ester, amide, prodrug or derivative of a compound as provided herein is a salt, ester, amide, prodrug or derivative that this not biologically or otherwise undesirable.
• Phenylalkyl means alkyl as defined above which is substituted with phenyl, e.g.,- CH 2 phenyl,-(CH 2 ) 2 Phenyl,-(CH 2 ) 3 phenyl, CH 3 CH(CH 3 )CH 2 phenyl, and the like and derivatives thereof. Phenylalkyl is a subset of the aralkyl group.
• phosphorylating activity is used herein to refer to the ability of a PKC to phosphorylate a hydroxyl group on a protein substrate, especially a hydroxyl group on a serine or threonine residue on a protein substrate.
• PKC overexpressing cell refers to a vertebrate cell, particularly a mammalian or avian cell, especially a mammalian cell, that expresses PKC, especially PKC- ⁇ , at a detectably greater level than a normal cell.
• the cell may be a vertebrate cell, such as a primate cell; an avian cell; a livestock animal cell such as a sheep cell, cow cell, horse cell, deer cell, donkey cell and pig cell ; a laboratory test animal cell such as a rabbit cell, mouse cell, rat cell, guinea pig cell and hamster cell; a companion animal cell such as a cat cell and dog cell; and a captive wild animal cell such as a fox cell, deer cell and dingo cell.
• the PKC overexpressing cell is a human cell.
• the PKC overexpressing cell is a cancer stem cell or a non-cancer stem cell tumor cell; preferably a cancer stem cell tumor cell. Overexpression can also be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a normal cell or comparison cell (e.g. a breast cell).
• polypeptide As used herein, the terms "polypeptide”, “proteinaceous molecule”, “peptide” and
• protein are used interchangeably to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers. These terms do not exclude modifications, for example, glycosylations, acetylations, phosphorylations and the like. Soluble forms of the subject proteinaceous molecules are particularly useful. Included within the definition are, for example, polypeptides containing one or more analogues of an amino acid including, for example, unnatural amino acids or polypeptides with substituted linkages.
• the terms “prevent”, “prevented” or “preventing”, refer to a prophylactic treatment which increases the resistance of a subject to developing the disease or condition or, in other words, decreases the likelihood that the subject will develop the disease or condition as well as a treatment after the disease or condition has begun in order to reduce or eliminate it altogether or prevent it from becoming worse. These terms also include within their scope preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it.
• pro-inflammatory immune response an immune response that includes one or more of increased levels of pro-inflammatory cytokines (e.g. IL-6, IL-23, IL-17, IL-la, IL- 1 ⁇ , and TNF-a) and/or increased levels of effector T cells, as compared to the levels of the one or more pro-inflammatory cytokines and/or the levels of effector T cells in a control, healthy mammal.
• pro-inflammatory cytokines e.g. IL-6, IL-23, IL-17, IL-la, IL- 1 ⁇ , and TNF-a
• the reduction may be determined subjectively, for example when a patient refers to their subjective perception of disease symptoms, such as pain, fatigue, etc.
• the reduction may be determined objectively, for example when the number of CSCs and/or non-CSC tumor cells in a sample from a patient is lower than in an earlier sample from the patient.
• the quantity of substance and/or phenomenon in the first sample is at least 10% lower than the quantity of the same substance and/or phenomenon in a second sample. In another embodiment, the quantity of the substance and/or phenomenon in the first sample is at least 25% lower than the quantity of the same substance and/or phenomenon in a second sample. In yet another embodiment, the quantity of the substance and/or phenomenon in the first sample is at least 50% lower than the quantity of the same substance and/or phenomenon in a second sample. In a further embodiment, the quantity of the substance and/or phenomenon in the first sample is at least 75% lower than the quantity of the same substance and/or phenomenon in a second sample. In yet another embodiment, the quantity of the substance and/or phenomenon in the first sample is at least 90% lower than the quantity of the same substance and/or phenomenon in a second sample.
• a difference may be expressed as an "n-fold" difference.
• an LSDl selective inhibitor is an agent which inhibits LSDl without substantially inhibiting the function of another LSD or another enzyme such as a monoamine oxidase (MAO).
• MAO monoamine oxidase
• an agent that is selective for LSDl exhibits LSDl selectivity of greater than about 2-fold, 5-fold, 10- fold, 20-fold, 50-fold or greater than about 100-fold with respect to inhibition of another LSD or MAO.
• selective molecules display at least 50-fold greater inhibition towards LSDl than another LSD or MAO.
• selective molecules display at least 100- fold greater inhibition towards LSDl than towards another LSD or MAO.
• selective molecules display at least 500-fold greater inhibition towards LSDl than towards another LSD or MAO. In yet further embodiments, selective molecules display at least 100-fold greater inhibition towards LSD1 than towards another LSD or MAO.
• salts and “prodrugs” include any pharmaceutically acceptable salt, ester, hydrate or any other compound which, upon administration to the recipient, is capable of providing (directly or indirectly) a proteinaceous molecule of the invention, or an active metabolite or residue thereof.
• Suitable pharmaceutically acceptable salts include salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulfonic, toluenesulfonic, benzenesulfonic, salicylic, sulfanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.
• pharmaceutically acceptable inorganic acids such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulf
• Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.
• basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl and diethyl sulfate; and others.
• non-pharmaceutically acceptable salts also fall within the scope of the invention since these may be useful in the preparation of pharmaceutically acceptable salts.
• the preparation of salts and prodrugs can be carried out by methods known in the art.
• metal salts can be prepared by reaction of a compound of the invention with a metal hydroxide.
• An acid salt can be prepared by reacting an appropriate acid with a proteinaceous molecule of the invention.
• stringency refers to the temperature and ionic strength conditions, and presence or absence of certain organic solvents during hybridization and washing procedures. The higher the stringency, the higher will be the degree of complementarity between immobilized target nucleotide sequences and the labelled probe polynucleotide sequences that remain hybridized to the target after washing.
• high stringency refers to temperature and ionic conditions under which only nucleotide sequences having a high frequency of
• the stringency required is nucleotide sequence dependent and depends upon the various components present during hybridization. Generally, stringent conditions are selected to be about 10 to 20°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
• T m is the temperature (under defined ionic strength and pH) at which 50% of a target sequence hybridizes to a complementary probe.
• subject refers to a vertebrate subject, particularly a mammalian or avian subject, for whom therapy or prophylaxis is desired. Suitable subjects include, but are not limited to, primates; avians; livestock animals such as sheep, cows, horses, deer, donkeys and pigs; laboratory test animals such as rabbits, mice, rats, guinea pigs and hamsters; companion animals such as cats and dogs; and captive wild animals such as foxes, deer and dingoes. In particular, the subject is a human. However, it will be understood that the aforementioned terms do not imply that symptoms are present.
• treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
• the effect may be therapeutic in terms of a partial or complete cure for a disease or condition (e.g. a hematologic malignancy) and/or adverse affect attributable to the disease or condition.
• a disease or condition e.g. a hematologic malignancy
• adverse affect attributable to the disease or condition e.g. a hematologic malignancy
• These terms also cover any treatment of a condition or disease in a mammal, particularly in a human, and include : (a) inhibiting the disease or condition, i.e., arresting its development; or (b) relieving the disease or condition, i.e., causing regression of the disease or condition.
• tumor refers to any neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
• cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized in part by unregulated cell growth.
• cancer refers to non-metastatic and metastatic cancers, including early stage and late stage cancers.
• precancerous refers to a condition or a growth that typically precedes or develops into a cancer.
• non-metastatic refers to a cancer that is benign or that remains at the primary site and has not penetrated into the lymphatic or blood vessel system or to tissues other than the primary site.
• a non-metastatic cancer is any cancer that is a Stage 0, I or II cancer.
• head stage cancer is meant a cancer that is not invasive or metastatic or is classified as a Stage 0, I or II cancer.
• late stage cancer generally refers to a Stage III or IV cancer, but can also refer to a Stage II cancer or a substage of a Stage II cancer.
• One skilled in the art will appreciate that the classification of a Stage II cancer as either an early stage cancer or a late stage cancer depends on the particular type of cancer.
• cancer examples include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, pancreatic cancer, colorectal cancer, lung cancer, hepatocellular cancer, gastric cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, retinoblastoma, melanoma, brain cancer, non-small cell lung cancer, squamous cell cancer of the head and neck, endometrial cancer, multiple myeloma, mesothelioma, rectal cancer and esophageal cancer.
• the cancer is breast cancer.
• the term "vector” refers to a polynucleotide molecule, suitably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned.
• a vector may contain one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector can be an autonomously replicating vector, i.e.
• a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication e.g. a linear or closed circular plasmid, an extra- chromosomal element, a mini-chromosome or an artificial chromosome.
• the vector can contain any means for assuring self-replication.
• the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
• a vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
• the vector is preferably a viral or viral-derived vector, which is operably functional in fungi, bacterial or animal cells, preferably mammalian cells.
• Such vector may be derived from a poxvirus, an adenovirus or yeast.
• the vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are known to those of skill in the art and include the nptll gene that confers resistance to the antibiotics kanamycin and G418 (Geneticin®) and the hph gene which confers resistance to the antibiotic hygromycin B.
• the present invention is based, in part, on the determination that inhibitors of LSDl inhibit immune checkpoints, particularly PD-L1 and/or PD-L2. Accordingly, the inventors have conceived that LSDl inhibitors may be used for a range of applications, including for enhancing an immune response in a subject to a target antigen by an immune-modulating agent or for the treatment of a cancer or an infection.
• the LSDl inhibitor includes and encompasses any active agent that reduces the accumulation, function or stability of LSDl ; or decreases expression of the LSDl gene; and such inhibitors include without limitation, small molecules and macromolecules such as nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, polysaccharides, lipopolysaccharides, lipids or other organic (carbon containing) or inorganic molecules.
• the LSDl inhibitor is an inhibitor of the catalytic activity of LSDl or an inhibitor of the nuclear translocation of LSDl.
• the LSDl inhibitor selectively inhibits LSDl over at least one other LSD or another enzyme such as an MAO. In some embodiments, the LSDl inhibitor selectively inhibits LSDl over the other LSD subtypes and MAOs. In some embodiments, the LSDl inhibitor exhibits LSDl selectivity of greater than about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or greater than about 100-fold with respect to inhibition of another LSD or MAO. In other
• selective molecules display at least 50-fold greater inhibition towards LSDl than another LSD or MAO. In further embodiments, selective molecules display at least 100-fold greater inhibition towards LSDl than towards another LSD or MAO. In still further embodiments, selective molecules display at least 500-fold greater inhibition towards LSDl than towards another LSD or MAO. In yet further embodiments, selective molecules display at least 100-fold greater inhibition towards LSDl than towards another LSD or MAO. In some embodiments, the LSDl inhibitor is a non-selective LSDl inhibitor.
• the LSDl inhibitor is an antagonistic nucleic acid molecule that functions to inhibit the transcription or translation of LSDl transcripts.
• Representative transcripts of this type include nucleotide sequences corresponding to any one the following sequences: (1) human LSDl nucleotide sequences as set forth for example in GenBank Accession Nos.
• nucleotide sequences that share at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity with any one of the sequences referred to in (1) ; (3) nucleotide sequences that hybridize under at least low, medium or high stringency conditions to the sequences referred to in (1) ; (4) nucleotide sequences that encode any one of the following amino acid sequences: human LSDl amino acid sequences as set forth for example in GenPept Accession Nos.
• Illustrative antagonist nucleic acid molecules include antisense molecules, aptamers, ribozymes and triplex forming molecules, RNAi and external guide sequences.
• the nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.
• Antagonist nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains.
• antagonist nucleic acid molecules can interact with LSDl mRNA or the genomic DNA of LSDl or they can interact with an LSDl polypeptide.
• antagonist nucleic acid molecules are designed to interact with other nucleic acids based on sequence homology between the target molecule and the antagonist nucleic acid molecule.
• the specific recognition between the antagonist nucleic acid molecule and the target molecule is not based on sequence homology between the antagonist nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place.
• anti-sense RNA or DNA molecules are used to directly block the translation of LSD 1 by binding to targeted mRNA and preventing protein translation.
• Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule may be designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule may be designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule.
• the antisense molecules bind the target molecule with a dissociation constant (K d ) less than or equal to 10 "6 , 10 "8 , 10 "10 , or 10 "12 .
• K d dissociation constant
• antisense oligodeoxyribonucleotides derived from the translation initiation site e.g. , between -10 and + 10 regions are employed.
• Aptamers are molecules that interact with a target molecule, suitably in a specific way.
• Aptamers are generally small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets.
• Aptamers can bind small molecules, such as ATP and theophylline, as well as large molecules, such as reverse transcriptase and thrombin.
• Aptamers can bind very tightly with K d s from the target molecule of less than 10 "12 M .
• the aptamers bind the target molecule with a K d less than 10 "6 , 10 "8 , 10 _1 °, or 10 "12 .
• Aptamers can bind the target molecule with a very high degree of specificity.
• aptamers have been isolated that have greater than a 10,000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule. It is desirable that an aptamer have a K d with the target molecule at least 10-, 100-, 1000-, 10,000-, or 100,000-fold lower than the K d with a background-binding molecule.
• a suitable method for generating an aptamer to a target of interest e.g. , PHD, FIH-1 or vHL is the
• SELEXTM Systematic Evolution of Ligands by Exponential Enrichment
• the SELEXTM method is described in U.S. Pat. No. 5,475,096 and U.S. Pat. No. 5,270,163 (see also WO 91/19813). Briefly, a mixture of nucleic acids is contacted with the target molecule under conditions favorable for binding. The unbound nucleic acids are partitioned from the bound nucleic acids, and the nucleic acid-target complexes are dissociated.
• the dissociated nucleic acids are amplified to yield a ligand-enriched mixture of nucleic acids, which is subjected to repeated cycles of binding, partitioning, dissociating and amplifying as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
• anti-LSDJ ribozymes are used for catalyzing the specific cleavage of LSD 1 RNA.
• the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage.
• ribozymes that catalyze nuclease or nucleic acid polymerase type reactions, which are based on ribozymes found in natural systems, such as hammerhead ribozymes, hairpin ribozymes, and tetrahymena ribozymes.
• ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo. Representative ribozymes cleave RNA or DNA substrates.
• ribozymes that cleave RNA substrates are employed.
• Specific ribozyme cleavage sites within potential RNA targets are initially identified by scanning the target molecule for ribozyme cleavage sites, which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
• Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acid.
• triplex molecules When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependent on both Watson-Crick and Hoogsteen base pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is generally desirable that the triplex forming molecules bind the target molecule with a K d less than 10 "6 , 10 "8 , 10 _1 °, or 10 "12 .
• EGSs External guide sequences
• RNAse P cleaves the target molecule.
• EGSs can be designed to specifically target a RNA molecule of choice.
• RNAse P aids in processing transfer RNA (tRNA) within a cell.
• Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA: EGS complex to mimic the natural tRNA substrate.
• EGS/RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukaryotic cells.
• RNA molecules that mediate RNA interference (RNAi) of an LSD1 gene or LSD1 transcript can be used to reduce or abrogate gene expression.
• RNAi refers to interference with or destruction of the product of a target gene by introducing a single-stranded or usually a double-stranded RNA (dsRNA) that is homologous to the transcript of a target gene.
• dsRNAi methods including double-stranded RNA interference (dsRNAi) or small interfering RNA (siRNA), have been extensively documented in a number of organisms, including mammalian cells and the nematode C. elegans (Fire et al. (1998) Nature, 391 : 806-811).
• RNAi can be triggered by 21- to 23-nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu et al. (2002) Mol. Cell. , 10 : 549-561; Elbashir et al. (2001) Nature, 411 : 494-498), or by micro-RNAs (miRNA), functional small-hairpin RNA (shRNA), or other dsRNAs which are expressed in vivo using DNA templates with RNA polymerase III promoters (Zeng et al. (2002) Mol. Cell, 9 : 1327-1333;
• dsRNA per se and especially dsRNA-producing constructs corresponding to at least a portion of an LSD1 gene are used to reduce or abrogate its expression.
• RNAi-mediated inhibition of gene expression may be accomplished using any of the techniques reported in the art, for instance by transfecting a nucleic acid construct encoding a stem-loop or hairpin RNA structure into the genome of the target cell, or by expressing a transfected nucleic acid construct having homology for an LSD1 gene from between convergent promoters, or as a head to head or tail to tail duplication from behind a single promoter.
• Any similar construct may be used so long as it produces a single RNA having the ability to fold back on itself and produce a dsRNA, or so long as it produces two separate RNA transcripts, which then anneal to form a dsRNA having homology to a target gene.
• RNAi-encoding nucleic acids can vary in the level of homology they contain toward the target gene transcript, i.e. , with dsRNAs of 100 to 200 base pairs having at least about 85% homology with the target gene, and longer dsRNAs, i.e., 300 to 100 base pairs, having at least about 75% homology to the target gene.
• RNA-encoding constructs that express a single RNA transcript designed to anneal to a separately expressed RNA, or single constructs expressing separate transcripts from convergent promoters are suitably at least about 100 nucleotides in length.
• RNA-encoding constructs that express a single RNA designed to form a dsRNA via internal folding are usually at least about 200 nucleotides in length.
• the promoter used to express the dsRNA-forming construct may be any type of promoter if the resulting dsRNA is specific for a gene product in the cell lineage targeted for destruction.
• the promoter may be lineage specific in that it is only expressed in cells of a particular development lineage. This might be advantageous where some overlap in homology is observed with a gene that is expressed in a non-targeted cell lineage.
• the promoter may also be inducible by externally controlled factors, or by intracellular environmental factors.
• RNA molecules of about 21 to about 23 nucleotides which direct cleavage of specific mRNA to which they correspond, as for example described by Tuschl ef al. in U.S. 2002/0086356, can be utilized for mediating RNAi.
• Such 21- to 23-nt RNA molecules can comprise a 3' hydroxyl group, can be single-stranded or double-stranded (as two 21- to 23-nt RNAs) wherein the dsRNA molecules can be blunt ended or comprise overhanging ends (e.g. 5', 3').
• the antagonist nucleic acid molecule is a siRNA.
• siRNAs can be prepared by any suitable method. For example, reference may be made to International Publication WO 02/44321, which discloses siRNAs capable of sequence-specific degradation of target mRNAs when base-paired with 3' overhanging ends, which is incorporated by reference herein. Sequence specific gene silencing can be achieved in mammalian cells using synthetic, short double-stranded RNAs that mimic the siRNAs produced by the enzyme dicer. siRNA can be chemically or in vitro- synthesized or can be the result of short double-stranded hairpin-like RNAs (shRNAs) that are processed into siRNAs inside the cell.
• shRNAs short double-stranded hairpin-like RNAs
• Synthetic siRNAs are generally designed using algorithms and a conventional DNA/RNA synthesizer.
• Suppliers include Ambion (Austin, Tex.), ChemGenes (Ashland, Mass.), Dharmacon (Lafayette, Colo.), Glen Research (Sterling, Va.), MWB Biotech (Esbersberg, Germany), Proligo (Boulder, Colo.), and Qiagen (Vento, The Netherlands).
• siRNA can also be synthesized in vitro using kits such as Ambion's SILENCERTM siRNA Construction Kit.
• siRNAs short hairpin RNAs
• Kits for the production of vectors comprising shRNA are available, such as, for example, Imgenex's GENESUPPRESSORTM Construction Kits and Invitrogen's BLOCK-ITTM inducible RNAi plasmid and lentivirus vectors.
• methods for formulation and delivery of siRNAs to a subject are also well known in the art.
• RNAi molecules e.g. LSDl siRNA and shRNA
• LSDl siRNA and shRNA are described in the art (e.g. Yang et a/. (2010) Proc. Natl. Acad. Sci. USA, 107 : 21499-21504; and He et al. (2012)
• the present invention further contemplates peptide or polypeptide based inhibitor compounds.
• BHC80 also known as PHD finger protein 21A
• the present invention further contemplates the use of BHC80 or biologically active fragments thereof for inhibiting LSDl catalytic activity.
• Amino acid sequences of BHC80 polypeptides, and nucleotide sequences encoding BHC80 polypeptides are publicly available. In this regard, reference may be made for example to GenBank Accession No. NP057705 for a Homo sapiens BHC80 amino acid sequence; and GenBank NM016621 for a nucleotide sequence encoding the amino acid sequence set forth in GenBank
• Illustrative BHC80 polypeptides are selected from the group consisting of: (1) a polypeptide comprising an amino acid sequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity with the amino acid sequence listed in any one of the GenBank BHC80 polypeptide entries noted above; (2) a polypeptide comprising an amino acid sequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity with the amino acid sequence listed in
• a BHC80 polypeptide can be introduced into a cell by delivering a polypeptide per se, or by introducing into the cell a BHC80 nucleic acid comprising a nucleotide sequence encoding a BHC80 polypeptide.
• a BHC80 nucleic acid comprises a nucleotide sequence selected from: (1) a BHC80 nucleotide sequence listed in any one of the GenBank BHC80 polynucleotide entries noted above; (2) a nucleotide sequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity with any one of the sequences referred to in (1) ; (3) a nucleotide sequence that hybridizes under at least low, medium or high stringency conditions to the sequences referred to in (1) ; (4) a nucleotide sequence that encodes an amino acid sequence listed in any one of the GenBank BHC80 polypeptide entries noted above; (5) a nucleotide sequence that encodes an
• the BHC80 nucleic acid can be in the form of a recombinant expression vector.
• the BHC80 nucleotide sequence can be operably linked to a transcriptional control element(s), e.g. a promoter, in the expression vector.
• Suitable vectors include, for example, recombinant
• retroviruses lentiviruses, and adenoviruses
• retroviral expression vectors lentiviral expression vectors
• nucleic acid expression vectors and plasmid expression vectors.
• the expression vector is integrated into the genome of a cell. In other cases, the expression vector persists in an episomal state in a cell.
• Suitable expression vectors include, but are not limited to, viral vectors (e.g. , viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g. , Li et a/. (1994) Invest
• the present invention also contemplates small molecule agents that reduce the catalytic activity of LSD1.
• Small molecule agents that reduce the catalytic activity of LSD1 that are suitable for use in the present invention include MAO inhibitors that also inhibit LSD1 catalytic activity; polyamine compounds that inhibit LSD1 catalytic activity; phenylcyclopropylamine derivatives that inhibit LSD1 catalytic activity; and the like.
• MAO inhibitors include MAO-A-selective inhibitors, MAO-B- selective inhibitors, and MAO non-selective inhibitors.
• Illustrative examples of MAO inhibitors include reported inhibitors of the MAO-A isoform, which preferentially deaminates 5- hydroxytry pta mine (serotonin) (5-HT) and norepinephrine (NE), and/or the MAO-B isoform, which preferentially deaminates phenylethylamine (PEA) and benzylamine (both MAO-A and MAO-B metabolize Dopamine (DA)).
• MAO inhibitors may be irreversible or reversible (e.g. reversible inhibitors of MAO-A (RIMA)), and may have varying potencies against MAO-A and/or MAO-B (e.g. non-selective dual inhibitors or isoform-selective inhibitors).
• RIMA reversible inhibitors of MAO-A
• MAO-B e.g. non-selective dual inhibitors or isoform-selective inhibitors.
• the MAO inhibitors are selected from : clorgyline; L-deprenyl; isocarboxazid (MarplanTM) ; ayahuasca ; nialamide; iproniazide; iproclozide; moclobemide
• rasagiline (AZILECT) ; bifemelane; desoxypeganine; harmine (also known as telepathine or banasterine) ; linezolid (ZYVOX, ZYVOXID) ; pargyline (EUDATIN, SUPIRDYL) ; dienolide kavapyrone desmethoxyyangonin; 5-(4-Arylmethoxyphenyl)-2-(2-cyanoethyl)tetrazoles; NCD36 (2-[/V-(4- phenylbenzenecarbonyl)]amino-6-(trans-2-phenylcyclopropan-l-amino)-/V-(3- methylbenzyl)hexanamide hydrochloride; and the like.
• Small molecule LSD1 inhibitors may also be selected from polyamine compounds as described for example by Woster ef al. in U.S. Publication No. 2007/0208082, which is incorporated herein by reference in its entirety.
• Illustrative polyamine inhibitors of LSD1 include compounds according to formula (II):
• each R 1 is independently selected from the group consisting of C 1- C 8 alkyl, C 4 -C 15 cycloalkyi, C3-C15 branched alkyl, C 6 -C 2 oaryl, C 6 -C 2 o heteroaryl, C 7 - C 24 aralkyl, C 7 -C 24 heteroaralkyl, and
• R 3 is selected from the group consisting of C 1- C 8 alkyl, C 4 -C 15 cycloalkyi, C3-C15 branched alkyl, C 6 -C 20 aryl, C 6 -C 20 heteroaryl, C 7 -C 24 aralkyl and C 7 -C 24 heteroaralkyl;
• each R 2 is independently selected from hydrogen or a C 1- C 8 alkyl.
• a suitable polyamine compound is a compound of Formula (II), wherein one or both Rl is a C6-C20 aryl, such as a single ring aryl, including without limitation, a phenyl.
• the compound is of the formula (II) and each Rl is phenyl.
• q is I, m and p are 3, and n is 4.
• q is I, m and p are 3, and n is 7.
• a suitable polyamine compound is a compound of Formula (II), where at least one or both R 1 is a C 8 -C 12 or a C 1- C 8 alkyl, such as a linear alkyl.
• R 1 may be a C 1- C 8 linear alkyl, such as methyl or ethyl.
• each R 1 is methyl.
• R 1 may comprise or be a C 4 -C 15 cycloalkyi group, such as a cycloalkyi group containing a linear alkyl group, where the cycloalkyi group is connected to the molecule either via its alkyl or cycloalkyi moiety.
• R 1 may be cyclopropylmethyl or cyclohexylmethyl.
• one R 1 is cyclopropylmethyl or cyclohexylmethyl and the other R 1 is a linear alkyl group, such as a linear C 1- C 8 unsubstituted alkyl group, including without limitation an ethyl group.
• R 1 is a C3-C15 branched alkyl group such as isopropyl.
• R 1 is a C 1- C 8 substituted alkyl
• the substituted alkyl may be substituted with any substituent, including a primary, secondary, tertiary or quaternary amine.
• R 1 is a C 1- C 8 alkyl group substituted with an amine such that R 1 may be e.g., alkyl-NH 2 or an alkyl-amine-alkyl moiety such as -(CH 2 )yNH(CH 2 ) z CH 3 where y and z are independently an integer from 1 to 8. In one embodiment, R 1 is -(CH 2 ) 3 NH 2 .
• the compound is of the formula (II) where one or both Ri is a C 7 - C 2 4 substituted or unsubstituted aralkyl, which in one embodiment is an aralkyl connected to the molecule via its alkyl moiety (e.g., benzyl). In one embodiment, both are aralkyl moieties wherein the alkyl portion of the moiety is substituted with two aryl groups and the moiety is connected to the molecule via its alkyl group.
• Ri is a C 7 - C 2 4 substituted or unsubstituted aralkyl, which in one embodiment is an aralkyl connected to the molecule via its alkyl moiety (e.g., benzyl).
• alkyl moiety e.g., benzyl
• both are aralkyl moieties wherein the alkyl portion of the moiety is substituted with two aryl groups and the moiety is connected to the molecule via its alkyl group.
• one or both is a C7-C24 aralkyl wherein the alkyl portion is substituted with two phenyl groups, such as when R 1 is 2,2-diphenylethyl or 2,2-dibenzylethyl.
• both of Formula III is 2,2- diphenylethyl and n is 1, 2 or 5.
• each of Formula III is 2,2-diphenylethyl, n is 1, 2 or 5 and m and p are each 1.
• At least one R 1 is hydrogen.
• the other R 1 may be any moiety listed above for R 1 , including an aryl group such as benzyl.
• Any of the compounds of Formula III listed above include compounds where at least one or both of R 2 is hydrogen or a C 1- C 8 substituted or unsubstituted alkyl.
• each R 2 is an unsubstituted alkyl such as methyl.
• each R 2 is hydrogen.
• Any of the compounds of Formula III listed above may be compounds where q is 1 and m and p are the same. Accordingly, the polyaminoguanidines of Formula III may be symmetric with reference to the polyaminoguanidine core (e.g.
• the compounds of Formula III may be asymmetric, e.g., when q is 0. In one embodiment, m and p are 1. In one embodiment, q is 0. In one embodiment, n is an integer from 1 to 5.
• the compound is a polyaminobiguanide or /V-alkylated polyaminobiguanide.
• An /V-alkylated polyaminobiguanide intends a polyaminobiguanide where at least one imine nitrogen of at least one biguanide is alkylated.
• the compound is a polyaminobiguanide of the Formula III, or a salt, solvate, or hydrate thereof, where q is 1, and at least one or each R 1 is of the structure:
• each R 3 is independently selected from the group consisting of C 1- C 8 alkyl, C 6 -C 2 o aryl, C 6 -C 20 heteroaryl, C 7 -C 24 aralkyl, and C 7 -C 24 heteroaralkyl; and each R 2 is independently hydrogen or a C 1- C 8 alkyl.
• each R 3 is a C 1- C 8 alkyl.
• the alkyl may be substituted with any substituent, including a primary, secondary, tertiary or quaternary amine.
• R 3 is a C 1- C 8 alkyl group substituted with an amine such that R 3 may be e.g. alkyl-NH 2 or an alkyl-amine-alkyl moiety such as -(CH 2 )yNH(CH 2 ) z CH 3 where y and z are
• R 3 is -(CH 2 ) 3 NH 2 .
• R 3 may also be a C 4 - C15 cycloalkyl or a C 3 -C 15 branched alkyl.
• at least one or each R 3 is a C 6 -C 20 aryl.
• q is I, m and p are 3, and n is 4. In another embodiment, q is I, m and p are 3, and n is 7.
• the compound is a polyaminobiguanide of Formula III where at least one R 3 is a C7-C24 aralkyi, which in one embodiment is an aralkyi connected to the molecule via its alkyl moiety.
• each R 3 is an aralkyi moiety where the alkyl portion of the moiety is substituted with one or two aryl groups and the moiety is connected to the molecule via its alkyl moiety.
• at least one or each R 3 is an aralkyi where the alkyl portion is substituted with two phenyl or benzyl groups, such as when R 3 is 2,2-diphenylethyl or 2,2-dibenzylethyl.
• each R 3 is 2,2-diphenylethyl and n is 1, 2 or 5.
• each R 3 is 2,2-diphenylethyl and n is 1, 2 or 5 and m and p are each 1.
• any of the polyaminobiguanide compounds of Formula (III listed above include compounds where at least one or both of R 2 is hydrogen or a C 1- C 8 alkyl.
• each R 2 is an unsubstituted alkyl, such as methyl.
• each R 2 is a hydrogen.
• any of the polyaminobiguanide compounds of Formula III listed above include compounds where q is 1 and m and p are the same. Accordingly, the polyaminobiguanides of Formula III may be symmetric with reference to the polyaminobiguanide core. Alternatively, the compounds of Formula III may be asymmetric. In one embodiment, m and p are 1. In one embodiment, q is 0. In one embodiment, n is an integer from 1 to 5. In one embodiment, q, m and p are each 1 and n is 1, 2 or 5.
• each R 1 , R 2 , R 3 , m, n, p and q disclosed in reference to Formula III intends and includes all combinations thereof the same as if each and every combination of R 1 , R 2 , R 3 , m, n, p and q were specifically and individually listed.
• Representative compounds of the Formula III include, e.g.:
• n 1, 2 or 3;
• each L is independently a linker of from about 2 to 14 carbons in length, for example of about 2, 3, 4, 5, 6, 8, 10, 12 or 14 carbon atoms in length, where the linker backbone atoms may be saturated or unsaturated, usually not more than one, two, three, or four unsaturated atoms will be present in a tether backbone, where each of the backbone atoms may be substituted or unsubstituted (for example with a C 1- C 8 alkyl), where the linker backbone may include a cyclic group (for example, a cyclohex-l,3-diyl group where 3 atoms of the cycle are included in the backbone) ;
• each R 12 is independently selected from hydrogen and a C 1- C 8 alkyl
• each R 11 is independently selected from hydrogen, C 2 -C 8 alkenyl, C 1- C 8 alkyl or C 3 -C 8 branched alkyl (e.g., methyl, ethyl, tert-butyl, isopropyl, pentyl, cyclobutyl, cyclopropylmethyl, 3-methylbutyl, 2- ethyl butyl, 5-NH 2 -pent-l-yl, propyl- l-ylmethyl( phenyl) phosphinate,
• a primary, secondary, tertiary or quaternary amine such as 5-NH 2 -cycloheptyl, 3- NH 2 -cyclopentyl and the like
• a C 2 -C 8 alkanoyl e.g. , an alkanoyl substituted with a methyl and an alkylazide group
• each L is independently selected from: -CHR 13 -(CH 2 ) m -, - CHR 13 -(CH2)n-CHR 13 -, -(CH 2 ) m CHR 13 -, -CH 2 -A-CH 2 - and -(CH 2 ) P - where:
• n is an integer from 1 to 5;
• A is (CH 2 ) m , ethane-l, l-diyl or cyclohex-l,3-diyl;
• p is an integer from 2 to 14, such as 1, 2, 3, 4 or 5;
• n is an integer from 1 to 12;
• R 1 3 is a C 1- Cs alkyl.
• the alkyl portion of the aralkyl or heteroaralkyi moiety is connected to the molecule via its alkyl moiety.
• R 1 i may be an aralkyl moiety such as 2-phenylbenzyl, 4-phenylbenzyl, 3,3,- diphenylpropyl, 2-(2-phenylethyl)benzyl, 2-methyl-3-phenylbenzyl, 2-napthylethyl, 4- (pyrenyl)butyl, 2-(3-methylnapthyl)ethyl, 2-(l,2-dihydroacenaphth-4-yl)ethyl and the like.
• R 11 may be a heteroaralkyi moiety such as 3- (benzoimidazolyl) propanoyl, l-(benzoimidazolyl)methanoyl, 2-(benzoimidazolyl)ethanoyl, 2- (benzoimidazolyl)ethyl and the like.
• the compound of Formula IV comprises at least one moiety selected from the group consisting of t-butyl, isopropyl, 2-ethylbutyl, 1-methylpropyl, 1- methylbutyl, 3-butenyl, isopent-2-enyl, 2-methylpropan-3-olyl, ethylthiyi, phenylthiyi, propynoyi, l-methyl-lH-pyrrole-2-yl; trifluoromethyl, cyclopropanecarbaldehyde, halo-substituted phenyl, nitro-substituted phenyl, alkyl-substituted phenyl, 2,4,6-trimethylbenzyl, halo-5-substituted phenyl (such as para-(F 3 S)-phenyl, azido and 2-methylbutyl.
• t-butyl isopropyl, 2-ethylbutyl, 1-methylpropy
• each R 11 is independently selected from hydrogen, n-butyl, ethyl, cyclohexylmethyl, cyclopentylmethyl, cyclopropylmethyl,
• the polyamine compound is of the structure of Formula IV, where n is 3, such that the compound has a structure according to Formula V:
• L 1 , L 2 and L 3 are independently selected from -CHR 13 -(CH 2 ) m -, -CHR 13 -(CH 2 ) n -CHR 13 -, - (CH 2 ) m -CHR 13 -, -CH 2 -A-CH 2 - and -(CH 2 ) P -;
• the polyamine compound is of the structure of Formula V where: L 1 is -CHR 13 -(CH 2 ) m -; L 2 is -CHR 13 -(CH 2 ) n -CHR 13 -; and L 3 is -(CH 2 ) m -CHR 13 -; where R 11 , R 1 2, R 1 3, rn and n are as defined above.
• the polyamine compound is of the structure of Formula V where: L 1 , L 2 and L 3 are independently -CH 2 -A-CH 2 -; and R 12 is hydrogen; where R 11 and A are as defined above. In particular embodiments, at least one of an A and an R 11 comprises an alkenyl moiety.
• the polyamine compound is of the structure of Formula V where: L 1 , L 2 and L 3 are independently -(CH 2 ) P - where p is as defined above; and R 12 is hydrogen.
• L 1 and L 3 p is an integer from 3 to 7
• L 3 p is an integer from 3 to 14.
• the polyamine compound is of the structure of Formula V where: L 1 , and L 3 are independently -(CH 2 ) P -; L 2 is -CH 2 -A-CH 2 -; and R 12 is hydrogen; where R 12 , p and A are as defined above.
• p is an integer from 2 to 6
• L 3 A is (CH 2 ) X where x is an integer from 1 to 5, or cyclohex-l,3-diyl.
• the polyamine compound is of the structure of Formula IV, where n is 2, such that the compound has a structure according to Formula VI :
• L 1 and L 2 are independently selected from -CHR 13 -(CH 2 ) m -CHR 13 -(CH 2 ) n -CHR 13 -, -(CH 2 ) n , CHR 13 -, -CH 2 -A-CH 2 - and -(CH 2 ) P -;
• the polyamine compound is of the structure of Formula VI where: L 1 is -(CH 2 ) P -; and L 2 is -(CH 2 ) m -CHR 13 -; where R 13 , m and p are as defined above.
• L 1 p is an integer from 3 to 10
• L 2 n is an integer from 2 to 9.
• the polyamine compound is of the structure of Formula VI where: L 1 and L 2 are -(CH 2 ) P -; where p is as defined above. In particular embodiments, p is an integer from 3 to 7.
• the polyamine compound is of the structure of Formula IV, where n is 1, such that the compound has a structure according to Formula VII :
• L 1 is -(CH 2 ) P - where p is as defined above.
• p is an integer from 2 to 6.
• one R 11 is an amino-substituted cycloalkyi (e.g. a cycloalkyi group substituted with a primary, secondary, tertiary or quaternary amine) or a C 2 -C 8 alkanoyl (which alkanoyl may be substituted with one or more substituents such as a methyl or an alkylazide group) ; and the other R 11 is a C 1- C 8 alkyl or a C 7 -C 24 aralkyl.
• cycloalkyi e.g. a cycloalkyi group substituted with a primary, secondary, tertiary or quaternary amine
• a C 2 -C 8 alkanoyl which alkanoyl may be substituted with one or more substituents such as a methyl or an alkylazide group
• the other R 11 is a C 1- C 8 alkyl or a C 7 -C 24 aralkyl.
• Representative compounds of the Formula IIII include, e.g. :
• Phenylcyclopropylamine derivatives that are inhibitors of LSD1 include compounds represented by Formula VIII :
• each of R1-R5 is independently selected from H, halo, alkyl, alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl, -L-heterocyclyl, -L-carbocyclyl, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl, amino, alkylamino, aryl, arylalkyi, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl, hydroxyl, heteroaryloxy, heteroarylalkoxy, isocyanato, isothiocyanate, nitro, sulfinyl, sulfonyl, sulfonamide, thiocarbonyl, thiocyanato,
• R6 is H or alkyl
• R7 is H, alkyl, or cycloalkyl ;
• R8 is an -L-heterocyclyl wherein the ring or ring system of the -L-heterocyclyl has from 0 to 3 substituents selected from halo, alkyl, alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl, -L- heterocyclyl, -L-carbocyclyl, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl, amino, alkylamino, aryl, arylalkyi, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl, hydroxyl, heteroaryloxy, heteroarylalkoxy, isocyanato, isothiocyanate, nitro, sulfinyl,
• each L is independently selected from -(CH 2 ) n -(CH 2 ) n -, -(CH 2 )nNH(CH 2 )n-, -(CH 2 ) n O(CH 2 ) n - , and -(CH 2 ) n S(CH 2 ) n -, and where each n is independently chosen from 0, 1, 2, and 3;
• L is a covalent bond.
• R6 and R7 are hydro.
• one of R1-R5 is selected from -L-aryl, -L-heterocyclyl, and -L-carbocyclyl.
• a compound of the invention is of Formula VIII where:
• each R1-R5 is optionally substituted and independently chosen from -H, halo, alkyl, alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl, -L-heteroaryl, -L-heterocyclyl, -L-carbocyclyl, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl, amino, aryl, arylalkyi, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl, hydroxyl, heteroaryloxy, heteroarylalkoxy, isocyanato, isothiocyanato, nitro, sulfinyl, sulfonyl, sulfonamide, thiocarbonyl
• R6 is chosen from -H and alkyl
• R7 is chosen from -H, alkyl, and cycloalkyl
• Rx when present is chosen from -H, alkyl, alkynyl, alkenyl, -L-carbocyclyl, -L-aryl, and -L- heterocyclyl, all of which are optionally substituted (except -H) ;
• Ry when present is chosen from -H, alkyl, alkynyl, alkenyl, -L-carbocyclyl, -L-aryl, and -L- heterocyclyl, all of which are optionally substituted (except -H), where Rx and Ry may be cyclically linked ;
• optionally substituted refers to zero or 1 to 4 optional substituents independently chosen from acylamino, acyloxy, alkenyl, alkoxy, cycloalkoxy, alkyl, alkylthio, cycloalkylthio, alkynyl, amino, aryl, arylalkyi, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, carbocyclyl, cyano, cyanato, halo, haloalkyl, haloaryl, hydroxyl, heteroaryl, heteroaryloxy, heterocyclyl, heteroarylalkoxy, isocyanato, isothiocyanato, nitro, sulfinyl, sulfonyl, sulfonamide, thiocarbonyl, thiocyanato, trihalomethanesulfonamido,
• R8 is -CORz, such that the compound is of the following structure:
• R1-R7 are described above; and Rz is -L-heterocyclyl which is optionally substituted with from 1-4 optional substituents independently chosen from acylamino, acyloxy, alkenyl, alkoxy, cycloalkoxy, alkyl, alkylthio, cycloalkylthio, alkynyl, amino, aryl, arylalkyi, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, carbocyclyl, cyano, cyanato, halo, haloalkyl, haloaryl, hydroxyl, heteroaryl, heteroaryloxy, heterocyclyl, heteroarylalkoxy, isocyanato, isothiocyanato, nitro, sulfinyl, sulfonyl, sulfonamide, thiocarbonyl, thio
• n is independently chosen from 0, 1, 2, and 3.
• each L is independently chosen from -(CH 2 ) n - (CH 2 ) n - and -(CH 2 ) n -0-(CH 2 ) n where each n is independently chosen from 0, 1, 2, and 3.
• each L is chosen from a bond, -CH 2 -, -CH 2 CH 2 -, -OCH 2 - , -OCH 2 CH 2 -, -CH 2 OCH 2 -, -CH 2 CH 2 CH 2 -, -OCH 2 CH 2 CH 2 -, and -CH 2 OCH 2 CH 2 -.
• each L is chosen from a bond, -CH 2 -, -CH 2 CH 2 -, OCH 2 -, and
• L is chosen from a bond and -CH 2 -.
• Exemplary compounds of Formula VIII include:
• Exemplary compounds of Formula VIII include: /V-cyclopropyl-2- ⁇ [(trans)-2- phenylcyclopropyl]amino ⁇ acetamide; 2- ⁇ [(trans)-2-phenylcyclopropyl]amino acetamide; N- cyclopropyl-2- ⁇ [(trans)-2-phenylcyclopropyl]amino ⁇ propanamide; 2- ⁇ [(trans)-2- phenylcyclopropyl]amino ⁇ -/V-prop-2-ynylacetamide; /V-isopropyl-2- ⁇ [(trans)-2- phenylcyclopropyl]amino ⁇ acetamide; /V-(tert-butyl)-2- ⁇ [(trans)-2- phenylcyclopropyl]amino ⁇ acetamide; /V-(2-morpholin-4-yl-2-oxoethyl)-/V-[(trans)-2- phenylcycl
• LSD1 inhibitor compounds may be selected from selective LSD1 and LSD1/MAOB dual inhibitors disclosed, for example, in WO2010/043721
• Representative compounds of this type include phenylcyclopropylamine derivatives or homologs, illustrative examples of which include phenylcyclopropylamine with one or two substitutions on the amine group; phenylcyclopropylamine with zero, one or two substitutions on the amine group and one, two, three, four, or five substitution on the phenyl group;
• phenylcyclopropylamine with zero, one or two substitutions on the amine group wherein the phenyl group of PCPA is substituted with (exchanged for) another ring system chosen from aryl or heterocyclyl to give an aryl- or heteroaryl-cyclopropylamine having zero, one or two substituents on the amine group; phenylcyclopropylamine wherein the phenyl group of PCPA is substituted with (exchanged for) another ring system chosen from aryl or heterocyclyl to give an aryl- or heterocyclyl-cyclopropylamine wherein said aryl- or heterocyclyl-cyclopropylamine on said aryl or heterocyclyl moiety has zero, one or two substitutions on the amine group and one, two, three, four, or five substitution on the phenyl group; phenylcyclopropylamine with one, two, three, four, or five substitution on the phenyl group; or any of the above
• Non-limiting embodiments of phenylcyclopropylamine derivatives or analogs include “cyclopropylamine amide” derivatives and "cyclopropylamine” derivatives.
• Specific examples of "cyclopropylamine acetamide” derivatives include, but are not limited to : /V-cyclopropyl-2- ⁇ [(trans)-2-phenylcyclopropyl]amino ⁇ acetamide; 2- ⁇ [(trans)-2- phenylcyclopropyl]amino ⁇ acetamide; /V-cyclopropyl-2- ⁇ [(trans)-2- phenylcyclopropyl]amino ⁇ propanamide; 2- ⁇ [(trans)-2-phenylcyclopropyl]amino ⁇ -/V-prop-2- ynylacetamide; /V-isopropyl-2- ⁇ [(trans)-2-phenylcyclopropyl]amino ⁇ acetamide; /V-(tert-buty
• cyclopropylamine derivatives, include, but are not limited to : N- 4-fluorobenzyl-N- ⁇ (trans)-2-[4-(benzyloxy)phenyl]cyclopropyl ⁇ amine, /V-4-methoxybenzyl-/V- ⁇ (trans)-2-[4-(benzyloxy)phenyl]cyclopropyl ⁇ amine, /V-benzyl-/V- ⁇ (trans)-2-[4- (benzyloxy)phenyl]cyclopropyl ⁇ amine, /V-[(trans)-2-phenylcyclopropyl]amino-methyl)pyridin-3-ol, /V-[(trans)-2-phenylcyclopropyl]-/V-(3-methylpyridin-2-ylmethyl)amine, /V-[(trans)-2- phenylcyclopropyl]-/V-(4-chloropyridin-3-yl
• LSDl inhibitors are, e.g., phenelzine or pargyline (propargylamine) or a derivative or analog thereof. Derivatives and analogs of phenelzine and pargyline
• phenelzine or pargyline derivative or analog thereof has selective LSDl or dual LSD1/MAOB inhibitory activity as described herein.
• the phenelzine derivative or analog has one, two, three, four or five substituents on the phenyl group.
• the phenelzine derivative or analog has the phenyl group substituted with (exchanged for) an aryl or heterocyclyl group wherein said aryl or heterocyclyl group has zero, one, two, three, four or five substituents.
• the pargyline derivative or analog has one, two, three, four or five substituents on the phenyl group.
• the pargyline derivative or analog has the phenyl group substituted with (exchanged for) an aryl or heterocyclyl group wherein said aryl or heterocyclyl group has zero, one, two, three, four or five substituents.
• the present invention also contemplates tranylcypromine derivatives as described for example by Binda et al. (2010, J. Am. Chem. Soc , 132 : 6827-6833, which is hereby incorporated by reference herein in its entirety) as inhibitors of LSDl catalytic function.
• Non-limiting example of such compounds include :
• LSDl inhibitor compounds may be selected from tranylcypromine analogs described by Benelkebir ef a/. (2011, Bioorg. Med. Chem. 19(12) : 3709-16, which is hereby incorporated by reference herein in its entirety).
• Representative analogs of this type, including o-, m- and p-bromo analogues include: (1R,2S)-2-(4-bromophenyl)cyclopropanamine hydrochloride (Compound 4c), (1R,2S)-2-(3-bromophenyl)cyclopropanamine hydrochloride (Compound 4d), (1R,2S)-2-(2-bromophenyl)cyclopropanamine hydrochloride (Compound 4e), (1R,2S)-2-(biphenyl- 4-yl)cyclopropanamine hydrochloride (Compound 4f).
• Non- limiting compounds disclosed by Culhane et a/ include propargyl-Lys-4, /V-methylpropargyl-Lys-4 H3-21, c/ ' s-3-chloroallyl-Lys-4 H3-21, trans-3-chloroallyl-Lys-4 H3-21, exo-cyclopropyl-Lys-4 H3- 21, endo-cyclopropyl-Lys-4 H3-21, endo-dimethylcyclopropyl-Lys-4, hydrazino-Lys-4 H3-21 and hydrazino-Lys-4 H3-21.
• cyclopropylamine compounds that are useful for inhibiting LSDl include those disclosed by Fyfe et a/, in U.S. Publication No. 2013/0197013, which is incorporated herein by reference in its entirety.
• Illustrative cyclopropylamine inhibitors of LSDl, which are disclosed as being selective for inhibiting LSDl, include compounds according to Formula IX:
• X 1 and X 2 are independently C(R2) or N ;
• X 3 and X 4 when present, are independently C(R2) or N ;
• (G) is a cyclyl group (as shown in Formula IX, the cyclyl group (G) has n substituents (Rl)) ;
• each (Rl) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl, -Ll-cyclyl, -Ll-amino, -Ll- hydroxyl, amino, amido, nitro, halo, haloalkyi, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl;
• each (R2) is independently chosen from -H, alkyl, alkenyl, alkynyl, cyclyl, -Ll-cyclyl, -Ll-amino, - Ll-hydroxyl, amino, amido, nitro, halo, haloalkyi, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl, wherein each (R2) group has 1, 2, or 3 independently chosen optional substituents or two (R2) groups can be taken together to form a heterocyclyl or aryl group having 1, 2, or 3 independently chosen optional substituents, wherein said optional substituents are independently chosen from alkyl, alkanoyl, heteroalkyl, heterocyclyl, haloalkyi, cycloalkyl, carbocyclyl, arylalkoxy, heterocyclylalkoxy
• R3 is -H or a ( C 1 -C 6 )alkyl group
• each LI is independently alkylene or heteroalkylene
• n 0, 1, 2, 3, 4 or 5
• X 1 is CH or N ;
• (G) is a cyclyl group; each (Rl) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl, -Ll-cyclyl, -Ll-amino, -Ll- hydroxyl, amino, amido, nitro, halo, haloalkyi, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl;
• each (R2) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl, -Ll-cyclyl, -Ll-amino, -Ll- hydroxyl, amino, amido, nitro, halo, haloalkyi, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl, wherein each (R2) group has 1, 2, or 3 optional substituents, wherein said optional substituents are independently chosen from alkyl, alkanoyl, heteroalkyl, heterocyclyl, haloalkyi, cycloalkyl, carbocyclyl, arylalkoxy,
• heterocyclylalkoxy aryl, aryloxy, heterocyclyloxy, alkoxy, haloalkoxy, oxo, acyloxy, carbonyl, carboxyl, carboxamido, cyano, halogen, hydroxyl, amino, aminoalkyl, amidoalkyl, amido, nitro, thiol, alkylthio, arylthio, sulfonamide, sulfinyl, sulfonyl, urea, or carbamate;
• each LI is independently alkylene or heteroalkylene
• (G) is a cyclyl group
• each (Rl) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl, -Ll-cyclyl, -Ll-amino, -Ll- hydroxyl, amino, amido, nitro, halo, haloalkyi, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl;
• each (R2) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl, -Ll-cyclyl, -Ll-amino, -Ll- hydroxyl, amino, amido, nitro, halo, haloalkyi, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl, wherein each (R2) group has 0, 1, 2, or 3 optional substituents, wherein said optional substituents are independently chosen from alkyl, alkanoyl, heteroalkyl, heterocyclyl, haloalkyi, cycloalkyl, carbocyclyl, arylalkoxy,
• heterocyclylalkoxy aryl, aryloxy, heterocyclyloxy, alkoxy, haloalkoxy, oxo, acyloxy, carbonyl, carboxyl, carboxamido, cyano, halogen, hydroxyl, amino, aminoalkyl, amidoalkyl, amido, nitro, thiol, alkylthio, arylthio, sulfonamide, sulfinyl, sulfonyl, urea, or carbamate;
• each LI is independently alkylene or heteroalkylene; m is 0, 1, 2 or 3; and
• n 0, 1, 2, 3, 4 or 5; or an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.
• (G) is a cyclyl group; each (Rl) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl, -Ll- cyclyl, -Ll-amino, -Ll-hydroxyl, amino, amido, nitro, halo, haloalkyi, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl ;
• each (R2) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl, -Ll-cyclyl, -Ll-amino, -Ll- hydroxyl, amino, amido, nitro, halo, haloalkyi, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl, wherein each (R2) group has 1, 2, or 3 optional substituents, wherein said optional substituents are independently chosen from alkyl, alkanoyl, heteroalkyl, heterocyclyl, haloalkyi, cycloalkyl, carbocyclyl, arylalkoxy,
• heterocyclylalkoxy aryl, aryloxy, heterocyclyloxy, alkoxy, haloalkoxy, oxo, acyloxy, carbonyl, carboxyl, carboxamido, cyano, halogen, hydroxyl, amino, aminoalkyl, amidoalkyl, amido, nitro, thiol, alkylthio, arylthio, sulfonamide, sulfinyl, sulfonyl, urea, or carbamate;
• R3 is -H or a (C 1- C 6 )alkyl group; each LI is alkylene or heteroalkylene; and n is 0, 1, 2, 3, 4 or 5; or an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.
• X 1 , X 2 , X 3 and X 4 are independently CH or N, provided that at least one of X 1 , X 2 , X 3 and X 4 is N ;
• (G) is a cyclyl group; each (Rl) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl, -Ll- cyclyl, -Ll-amino, -Ll-hydroxyl, amino, amido, nitro, halo, haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl;
• each (R2) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl, -Ll-cyclyl, -Ll-amino, -Ll- hydroxyl, amino, amido, nitro, halo, haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl, wherein each (R2) group has 1, 2, or 3 optional substituents, wherein said optional substituents are independently chosen from alkyl, alkanoyl, heteroalkyl, heterocyclyl, haloalkyl, cycloalkyl, carbocyclyl, arylalkoxy,
• heterocyclylalkoxy aryl, aryloxy, heterocyclyloxy, alkoxy, haloalkoxy, oxo, acyloxy, carbonyl, carboxyl, carboxamido, cyano, halogen, hydroxyl, amino, aminoalkyl, amidoalkyl, amido, nitro, thiol, alkylthio, arylthio, sulfonamide, sulfinyl, sulfonyl, urea, or carbamate; each LI is alkylene or heteroalkylene;
• n 0, 1, 2, 3, 4 or 5;
• Representative compounds according to Formula IX are suitably selected from : (trans)- 2-(3'-(trifluoromethyl)biphenyl-4-yl)cyclopropanamine; (trans)-2-(terphenyl-4- yl)cyclopropanamine; 4'-((trans)-2-aminocyclopropyl)biphenyl-4-ol; 4'-((trans)-2- aminocyclopropyl)biphenyl-3-ol ; (trans) -2- (6-(3-(trif luoromethy I) phenyl) pyridin-3- yl)cyclopropanamine; (trans)-2-(6-(3,5-dichlorophenyl)pyridin-3-yl)cyclopropanamine; (trans) -2- (6-(4-chlorophenyl)pyridin-3-yl)cyclopropanamine; (trans)-2-(6-(3-chlorophenyl)
• LSD1 inhibitor compounds are selected from
• Ar! is a 5 to 7 membered aryl or heteroaryl ring
• Ar 2 and Ar 3 are each independently selected from a 5 to 7 membered aryl or heteroaryl ring, optionally substituted with 1 to 3 substituents;
• R 3 is selected from hydrogen, -C 1-6 alkyl or -OH; m is an integer from 1 to 5; and
• n is an integer from 1 to 3;
• Ar ! is a six membered aryl or heteroaryl ring, especially phenyl, pyridine, pyrimidine, pyrazine 1,3,5-triazine, 1,2,4-trazine and 1,2,3-triazine, more especially phenyl;
• Ar 2 is a six membered aryl or heteroaryl ring, especially phenyl, pyridine, pyrimidine, pyrazine 1,3,5-triazine, 1,2,4-trazine and 1,2,3-triazine, especially phenyl ; especially where the six membered aryl or heteroaryl ring is optionally substituted with one optional substituent, especially in the 3 or 4 position ;
• Ar 3 is a six membered aryl or heteroaryl ring, especially phenyl, pyridine, pyrimidine, pyrazine 1,3,5-triazine, 1,2,4-trazine and 1,2,3-triazine, especially phenyl ; especially where the six membered aryl or heteroaryl ring is optionally substituted with one optional substituent, especially in the 3 or 4 position.
• Particular optional substituents for An and Ar 2 include -C 1-6 alkyl, -C 2 -6alkenyl, -CH 2 F, -CHF 2 , -CF 3 , halo, aryl, heteroaryl, -C(0)NHC 1-6 alkyl, -C(0)NHC 1-6 alkylNH 2 , -C(0)-heterocyclyl, especially methyl, ethyl, propyl, butyl, t-butyl, -CH 2 F, -CHF 2 , -CH 3 , CI, F, phenyl, -C(0)NH(CH 2 )!_4NH 2 and - C(0)-heterocyclyl;
• R 3 is H, -C 1-3 alkyl or -OH, especially H, -CH 3 or -OH.
• n 2 to 5, especially 3 to 5, more especially 4,
• n 1 or 2, especially 1.
• the compounds of Formula XIV are compounds of Formula XlVa :
• Non-limiting compounds represented by Formula XIV include the following :
• LSDl inhibitors include, but are not limited to those, e.g. , disclosed in Ueda et a/. (2009, J. Am. Chem. Soc , 131(48) : 17536-17537) including ; M imasu et a/. (2010, Biochemistry, 49(30) : 6494-6503).
• the invention also encompasses LSDl inhibitors identified by any suitable screening assay. Accordingly, the present invention extends to methods of screening for inhibitory agents that are useful for inhibiting LSDl and, in turn, for inhibiting immune checkpoints, particularly PD-L1 and/or PD-L2.
• the screening methods comprise (1) contacting a preparation with a test agent, wherein the preparation comprises (i) a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of LSDl or to a variant or derivative thereof; or (ii) a polynucleotide comprising a nucleotide sequence from which a transcript of an LSDl gene or portion thereof is producible, or (iii) a polynucleotide comprising at least a portion of a genetic sequence (e.g.
• a transcriptional element that regulates the expression of an LSDl gene, which is operably linked to a reporter gene; and (2) detecting a change in the level or functional activity of the polypeptide, the polynucleotide or an expression product of the reporter gene, relative to a reference level or functional activity in the absence of the test agent.
• a detected reduction in the level and/or functional activity of the polypeptide, transcript or transcript portion or an expression product of the reporter gene, relative to a normal or reference level and/or functional activity in the absence of the test agent indicates that the agent is useful for inhibiting immune checkpoints, particularly PD-L1 and/or PD-L2.
• Inhibitors falling within the scope of the present invention include inhibitors of the level, functional activity or nuclear translocation of LSDl, including antagonistic antigen-binding molecules, and inhibitor peptide fragments, antisense molecules, ribozymes, RNAi molecules and co-suppression molecules as well as polysaccharide and lipopolysaccharide inhibitors of LSDl.
• Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons.
• Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, desirably at least two of the functional groups.
• the candidate agent often comprises homocyclic carbon or heterocyclic structures or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
• Candidate agents are also found among biomolecules including, but not limited to : peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogues or combinations thereof.
• Small (non-peptide) molecule inhibitors of LSDl are particularly advantageous.
• small molecules are desirable because such molecules are more readily absorbed after oral administration, have fewer potential antigenic determinants, or are more likely to cross the cell membrane than larger, protein-based pharmaceuticals.
• Small organic molecules may also have the ability to gain entry into an appropriate cell and affect the expression of a gene (e.g., by interacting with the regulatory region or transcription factors involved in gene expression) ; or affect the activity of a gene by inhibiting or enhancing the binding of accessory molecules.
• libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.
• natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries.
• Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogues.
• Screening may also be directed to known pharmacologically active compounds and chemical analogues thereof.
• Screening for modulatory agents according to the invention can be achieved by any suitable method.
• the method may include contacting a cell expressing a
• target molecules a downstream cellular target of the polypeptide or of the transcript
• Detecting such modulation can be achieved utilizing techniques including, but not restricted to, ELISA, cell-based ELISA, inhibition ELISA, Western blots, immunoprecipitation, slot or dot blot assays, immunostaining, RIA, scintillation proximity assays, fluorescent immunoassays using antigen-binding molecule conjugates or antigen conjugates of fluorescent substances such as fluorescein or rhodamine, Ouchterlony double diffusion analysis, immunoassays employing an avidin-biotin or a streptavidin-biotin detection system, and nucleic acid detection assays including reverse transcriptase polymerase chain reaction (RT-PCR).
• a polynucleotide from which LSD1 is regulated or expressed may be naturally occurring in the cell which is the subject of testing or it may have been introduced into the host cell for the purpose of testing.
• the naturally-occurring or introduced polynucleotide may be constitutively expressed, thereby providing a model useful in screening for agents which down-regulate expression of an encoded product of the sequence wherein the down regulation can be at the nucleic acid or expression product level.
• that polynucleotide may comprise the entire coding sequence that codes for LSD1 or it may comprise a portion of that coding sequence (e.g.
• the active site of LSD1) or a portion that regulates expression of the corresponding gene that encodes LSD1 e.g. an LSD1 promoter
• the promoter that is naturally associated with the polynucleotide may be introduced into the cell that is the subject of testing.
• detecting modulation of the promoter activity can be achieved, for example, by operably linking the promoter to a suitable reporter polynucleotide including, but not restricted to, green fluorescent protein (GFP), luciferase, ⁇ -galactosidase and catecholamine acetyl transferase (CAT). Modulation of expression may be determined by measuring the activity associated with the reporter polynucleotide.
• GFP green fluorescent protein
• CAT catecholamine acetyl transferase
• These methods provide a mechanism for performing high throughput screening of putative inhibitory agents such as proteinaceous or non-proteinaceous agents comprising synthetic, combinatorial, chemical and natural libraries. These methods will also facilitate the detection of agents which bind either the polynucleotide encoding LSD1 or which inhibit the expression of an upstream molecule, which subsequently inhibits the expression of the polynucleotide encoding LSD1. Accordingly, these methods provide a mechanism of detecting agents that either directly or indirectly inhibit the expression or activity of LSD1 according to the invention.
• test agents are screened using commercially available assays, illustrative examples of which include EpiQuik Histone Demethylase LSD1 Inhibitor Screening Assay Kit (Epigentek Group, Brooklyn, NY) or the LSD1 Inhibitor Screening Assay Kit (Cayman Chemical Company, Ann Arbor, MI).
• Compounds may be further tested in the animal models to identify those compounds having the most potent in vivo effects. These molecules may serve as "lead compounds" for the further development of pharmaceuticals by, for example, subjecting the compounds to sequential modifications, molecular modeling, and other routine procedures employed in rational drug design.
• the present inventors have also conceived novel proteinaceous molecules that inhibit LSD1.
• a proteinaceous molecule comprising, consisting or consisting essentially of a sequence corresponding to residues 108 to 118 of LSDl inhibits LSDl, especially the nuclear translocation of LSDl.
• Such proteinaceous molecules inhibit formation and maintenance of cancer stem cell and non-cancer stem cell tumor cells.
• the proteinaceous molecules of the invention may be used for the treatment or prevention of a cancer.
• the proteinaceous molecules of the invention may be useful in conditions involving PD-L1 and/or PD-L2 activity, such as an infection, or for enhancing an immune response.
• the LSDl inhibitor is an isolated or purified proteinaceous molecule comprising, consisting or consisting essentially of a sequence corresponding to residues 108 to 118 of LSDl.
• the proteinaceous molecule is an isolated or purified
• Zi and Z 2 are independently absent or are independently selected from at least one of a proteinaceous moiety comprising from about 1 to about 50 amino acid residues (and all integer residues in between), and a protecting moiety;
• Xi is selected from small amino acid residues, including S, T, A, G and modified forms thereof.
• "Xi" is selected from S and A.
• Xi is selected from S, A and modified forms thereof. In some embodiments, "Xi" is selected from S, A and S(P0 3 ).
• "Xi" is a modified form of S, especially S(P0 3 ).
• ⁇ ' is a proteinaceous molecule represented by Formula II:
• X 4 is selected from any amino acid residue.
• X 3 is selected from basic amino acid residues including R, K and modified forms thereof. In some embodiments, “X 3 " is R.
• X is selected from aromatic amino acid residues, including F, Y, W and modified forms thereof. In some embodiments, "X " is W.
• the isolated or purified proteinaceous molecule of Formula I comprises, consists or consists essentially of an amino acid sequence represented by SEQ ID NO : 1, 2 or 3 :
• RRTSRRKRAKV [SEQ ID NO: 1] ;
• RRTARRKRAKV [SEQ ID NO: 2] ;
• RW RRTARRKRAKV [SEQ ID NO: 3].
• the isolated or purified proteinaceous molecule of Formula I comprises, consists or consists essentially of an amino acid sequence represented by SEQ ID NO : 1 or 2.
• the isolated or purified proteinaceous molecule of Formula I is other than a proteinaceous molecule consisting of the amino acid sequence of SEQ ID NO : 4:
• the present invention also contemplates proteinaceous molecules that are variants of SEQ ID NO : 1, 2 or 3.
• variant proteinaceous molecules include proteins derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N- terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein ; or substitution of one or more amino acids at one or more sites in the native protein.
• Variant proteins encompassed by the present invention are biologically active, that is, they continue to possess the desired biological activity of the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation.
• the proteinaceous molecules of SEQ ID NO: 1, 2 and/or 3 may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
• amino acid sequence variants of SEQ ID NO : 1, 2 and/or 3 can be prepared by mutagenesis of nucleic acids encoding the amino acid sequence of SEQ ID NO: 1, 2 and/or 3. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82 : 488-492), Kunkel et al.
• Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with screening assays to identify active variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89 : 7811-7815; Delgrave et al. (1993) Protein Engineering, 6: 327-331). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be desirable as discussed in more detail below.
• Variant peptides or polypeptides of the invention may contain conservative amino acid substitutions at various locations along their sequence, as compared to a parent (e.g. naturally- occurring or reference) amino acid sequence, such as SEQ ID NO: 1, 2 and/or 3.
• a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art as discussed in detail below.
• amino acid sequence of the proteinaceous molecules of the invention is defined in terms of amino acids of certain characteristics or sub-classes. Amino acid residues are generally sub-classified into major sub-classes as follows:
• Acidic The residue has a negative charge due to loss of a proton at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
• Amino acids having an acidic side chain include glutamic amid and aspartic acid.
• Basic The residue has a positive charge due to association with protons at physiological pH or within one or two pH units thereof (e.g. histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
• Amino acids having a basic side chain include arginine, lysine and histidine.
• the residue is charged at physiological pH and, therefore, includes amino acids having acidic or basic side chains, such as glutamic acid, aspartic acid, arginine, lysine and histidine.
• Hydrophobic The residue is not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
• Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.
• Neutral/polar The residues are not charged at physiological pH but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
• Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.
• proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the a-amino group, as well as the a-carbon.
• amino acid similarity matrices e.g. PAM 120 matrix and PAM250 matrix as disclosed for example by Dayhoff ef al., (1978), A model of evolutionary change in proteins. Matrices for determining distance relationships In M. 0. Dayhoff, (ed.), Atlas of protein sequence and structure, Vol. 5, pp.
• proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a "small" amino acid.
• Amino acid residues can be further sub-classified as cyclic or non-cyclic, and aromatic or non-aromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large.
• the residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not.
• Small amino acid residues are, of course, always non-aromatic.
• amino acid residues may fall in two or more classes.
• sub-classification according to this scheme is presented in Table 1.
• Conservative amino acid substitution also includes groupings based on side chains.
• a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan ; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
• Amino acid substitutions falling within the scope of the invention are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity.
• amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains.
• the first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains;
• the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine;
• the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, Biochemistry, third edition, Wm.C. Brown Publishers (1993).
• a predicted non-essential amino acid residue in a peptide of the invention is typically replaced with another amino acid residue from the same side chain family.
• mutations can be introduced randomly along all or part of the coding sequence of a peptide of the invention, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide, as described for example herein, to identify mutants which retain that activity.
• the encoded peptide can be expressed recombinantly and its activity determined.
• non-essential amino acid residue is a residue that can be altered from the wild-type sequence of an embodiment peptide of the invention without abolishing or substantially altering one or more of its activities.
• the alteration does not substantially alter one of these activities, for example, the activity is at least 20%, 40%, 60%, 70% or 80% of that of the wild-type.
• an "essential" amino acid residue is a residue that, when altered from the wild-type sequence of an embodiment peptide of the invention, results in abolition of an activity of the parent molecule such that less than 20% of the wild-type activity is present.
• such essential amino acid residues include Arg (or modified form thereof) at position 2, Arg (or modified form thereof) at positions 2, 3, 6, 7 and 9, Thr (or modified form thereof) at position 4, Lys (or modified form thereof) at positions 8 and 11, Ala (or modified form thereof) at position 10, and Val (or modified form thereof) at position 12, relative to the numbering of Formula (I) commencing at Zi.
• the present invention also contemplates variants of the proteinaceous molecules of SEQ ID NO 1, 2 and/or 3 of the invention, wherein the variants are distinguished from the parent sequence by the addition, deletion, or substitution of one or more amino acid residues.
• variants will display at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity to a parent or reference proteinaceous molecule sequence as, for example, set forth in SEQ ID NO : 1, 2 or 3, as determined by sequence alignment programs described elsewhere herein using default parameters.
• variants will have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a parent or reference peptide sequence as, for example, set forth in SEQ ID NO : 1, 2 or 3, as determined by sequence alignment programs described herein using default parameters.
• Variants of SEQ ID NO : 1, 2 and/or 3, which fall within the scope of a variant peptide of the invention may differ from the parent molecule generally by at least 1, but by less than 4, 3, 2 or 1 amino acid residue(s).
• a variant peptide of the invention differs from the parent molecule generally by at least 1, but by less than 4, 3, 2 or 1 amino acid residue(s).
• the amino acid sequence of the variant peptide of the invention comprises Arg (or modified form thereof) at position 2, Arg (or modified form thereof) at positions 2, 3, 6, 7 and 9, Thr (or modified form thereof) at position 4, Lys (or modified form thereof) at positions 8 and 11, Ala (or modified form thereof) at position 10, and Val (or modified form thereof) at position 12, relative to the numbering of Formula (I) commencing at Zi.
• the amino acid sequence of the variant peptide of the invention comprises the proteinaceous molecule of Formula I.
• the variant peptide of the invention inhibits LSD1, particularly LSD1 nuclear translocation.
• sequences are typically aligned for maximum similarity or identity. "Looped" out sequences from deletions or insertions, or mismatches, are generally considered differences. The differences are, suitably, differences or changes at a non-essential residue or a conservative substitution.
• the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
• the length of a reference sequence aligned for comparison purposes is at least 40%, more usually at least 50% or 60%, and even more usually at least 70%, 80%, 90% or 100% of the length of the reference sequence.
• the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
• the percent identity between the two sequences is a function of the number of identical amino acid residues shared by the sequences at individual positions, taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
• the percent similarity between the two sequences is a function of the number of identical and similar amino acid residues shared by the sequences at individual positions, taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
• the percent identity or similarity between amino acid sequences is determined using the Needleman and WCinsch (1970, J. Mol. Biol., 48 : 444-453) algorithm which has been incorporated into the GAP program in the GCG software package (Devereaux, et al. (1984) Nucleic Acids Research, 12 : 387-395), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In some
• the percent identity or similarity between amino acid sequences can be determined using the algorithm of Meyers and Miller (1989, Cabios, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
• the present invention also contemplates an isolated, synthetic or recombinant peptide that is encoded by a polynucleotide sequence that hybridizes under stringency conditions as defined herein, especially under medium, high or very high stringency conditions, preferably under high or very high stringency conditions, to a polynucleotide sequence encoding the peptides of SEQ ID NO : 1, 2 and/or 3 or the non-coding strand thereof.
• the invention also contemplates an isolated nucleic acid molecule comprising a polynucleotide sequence that hybridizes under stringency conditions as defined herein, especially under medium, high or very high stringency conditions, preferably under high or very high stringency conditions, to a polynucleotide sequence encoding the peptides of SEQ ID NO : 1, 2 and/or 3 or the non-coding strand thereof.
• hybridizes under stringency conditions describes conditions for hybridization and washing and may encompass low stringency, medium stringency, high stringency and very high stringency conditions.
• Low stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP0 4 (pH 7.2), 7% sodium dodecyl sulfate (SDS) for hybridization at 65° C, and (i) 2 x sodium chloride/sodium citrate (SSC), 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP0 4 (pH 7.2), 5% SDS for washing at room temperature.
• BSA Bovine Serum Albumin
• 1 mM EDTA 1 M NaHP0 4 (pH 7.2)
• SDS sodium dodecyl sulfate
• SSC sodium chloride/sodium citrate
• low stringency conditions includes hybridization in 6 ⁇ SSC at about 45° C, followed by two washes in 0.2 x SSC, 0.1% SDS at least at 50° C (the temperature of the washes can be increased to 55° C for low stringency conditions).
• Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C, and at least about 0.1 M to at least about 0.2 M salt for washing at 55° C.
• Medium stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP0 4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 ⁇ SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP0 4 (pH 7.2), 5% SDS for washing at 60-65° C.
• BSA Bovine Serum Albumin
• 1 mM EDTA 1 mM EDTA, 0.5 M NaHP0 4 (pH 7.2), 7% SDS for hybridization at 65° C
• 2 ⁇ SSC 0.1% SDS
• BSA Bovine Serum Albumin
• High stringency conditions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from about 0.01 M to about 0.15 M salt for hybridization at 42° C, and about 0.01 M to about 0.02 M salt for washing at 55° C.
• High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHP0 4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 0.2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP0 4 (pH 7.2), 1% SDS for washing at a temperature in excess of 65° C.
• One embodiment of high stringency conditions includes hybridizing in 6 ⁇ SSC at about 45° C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 65° C.
• an isolated, synthetic or recombinant peptide of the invention that is encoded by a polynucleotide sequence that hybridizes under high stringency conditions to a polynucleotide sequence encoding the peptides of SEQ ID NO : 1, 2 and/or 3 or the non-coding strand thereof.
• the isolated, synthetic or recombinant peptide of the invention is encoded by a polynucleotide sequence that hybridizes under very high stringency conditions to a polynucleotide sequence encoding the peptides of SEQ ID NO: 1, 2 and/or 3 or the non-coding strand thereof.
• the amino acid sequence of the variant peptide of the invention comprises Arg (or modified form thereof) at position 2, Arg (or modified form thereof) at positions 2, 3, 6, 7 and 9, Thr (or modified form thereof) at position 4, Lys (or modified form thereof) at positions 8 and 11, Ala (or modified form thereof) at position 10, and Val (or modified form thereof) at position 12, relative to the numbering of Formula (I) commencing at Zi.
• the amino acid sequence of the variant peptide of the invention comprises the proteinaceous molecule of Formula I.
• the variant peptide of the invention inhibits LSD1, particularly LSD1 nuclear translocation.
• the T m of a duplex DNA decreases by approximately 1° C with every increase of 1% in the number of randomly mismatched base pairs. Washing is generally carried out at T m - 15° C for high stringency, or T m - 30° C for moderate stringency.
• a membrane e.g. a nitrocellulose membrane or a nylon membrane
• immobilized DNA is hybridized overnight at 42° C in a hybridization buffer (50% deionized formamide, 5 ⁇ SSC, 5 ⁇ Denhardt's solution (0.1% ficoll, 0.1% polyvinylpyrrolidone and 0.1% BSA), 0.1% SDS and 200 mg/mL denatured salmon sperm DNA) containing labeled probe.
• the membrane is then subjected to two sequential medium stringency washes (i.e.
• the proteinaceous molecules of the present invention also encompass peptides comprising amino acids with modified side chains, incorporation of unnatural amino acid residues and/or their derivatives during peptide synthesis and the use of cross-linkers and other methods which impose conformational constraints on the peptides of the invention.
• side chain modifications include modifications of amino groups, such as by acylation with acetic anhydride; acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; amidination with methylacetimidate; carbamoylation of amino groups with cyanate; pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with sodium borohydride; reductive alkylation by reaction with an aldehyde followed by reduction with sodium borohydride; and trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulfonic acid (TNBS).
• modifications of amino groups such as by acylation with acetic anhydride; acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; amidination with methylacetimidate; carbamoylation of amino groups with cyanate; pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with sodium
• the carboxyl group may be modified by carbodiimide activation through O-acylisourea formation followed by subsequent derivatization, for example, to a corresponding amide.
• the guanidine group of arginine residues may be modified by formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
• Tryptophan residues may be modified, for example, by alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfonyl halides, or by oxidation with /V-bromosuccinimide.
• Tyrosine residues may be modified by nitration with tetranitromethane to form 3- nitrotyrosine derivatives.
• Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of 4-amino butyric acid, 6-aminohexanoic acid, 4- amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, t- butylglycine, norleucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine, selenocysteine, O-phosphoserine, and ⁇ , ⁇ -difluoromethylenephosphonoserine and/or D-isomers of amino acids.
• Table 3 Exemplary Unnatural Amino Acids
• the proteinaceous molecules of the invention may inherently permeate membranes, membrane permeation may further be increased by the conjugation of a membrane permeating moiety to the proteinaceous molecule. Accordingly, in some embodiments the proteinaceous molecules of the invention comprise at least one membrane permeating moiety.
• the membrane permeating moiety may be conjugated at any point of the proteinaceous molecule.
• Suitable membrane permeating moieties include lipid moieties, cholesterol and proteins, such as cell-penetrating peptides and polycationic peptides; especially lipid moieties.
• Suitable cell penetrating peptides may include the peptides described in, for example, US 20090047272, US 20150266935 and US 20130136742. Accordingly, suitable cell penetrating peptides may include, but are not limited to, basic poly(Arg) and poly(Lys) peptides and basic poly(Arg) and poly(Lys) peptides containing non-natural analogues of Arg and Lys residues such as YGRKKRPQRRR (HIV TAT47-57; SEQ ID NO: 6), RRWRRWWRRWRRWRR (W/R; SEQ ID NO: 7), CWK 18 (AlkCWK 18 ; SEQ ID NO: 8), K 18 WCCWK 18 (Di-CWK 18 ; SEQ ID NO: 9),
• WTLNSAGYLLGKINLKALAALAKKIL Transportan; SEQ ID NO: 10), GLFEALEELWEAK (DipaLytic; SEQ ID NO: 11), K 16 GGCRGDMFGCAKi 6 RGD (K 16 RGD; SEQ ID NO: 12), K 16 GGCMFGCGG (PI ; SEQ ID NO: 13), K 16 ICRRARGDNPDDRCT (P2; SEQ ID NO: 14), KKWKMRRNQFWVKVQRbAK (B) bA (P3; SEQ ID NO: 15), VAYISRGGVSTYYSDTVKGRFTRQKYNKRA (P3a; SEQ ID NO: 16),
• IGRIDPANGKTKYAPKFQDKATRSNYYGNSPS P9.3; SEQ ID NO: 17), KETWWETWWTEWSQPKKKRKV (Pep-1 ; SEQ ID NO: 18), PLAEIDGIELTY (Plae; SEQ ID NO: 19), K 16 GGPLAEIDGIELGA (Kplae; SEQ ID NO: 20), K 15 GGPLAEIDGIELCA (cKplae; SEQ ID NO: 21), GALFLGFLGGAAGSTMGAWSQPKSKRKV (MGP; SEQ ID NO: 22), WEAK(LAKA) 2 -LAKH(LAKA) 2 LKAC (HA2; SEQ ID NO: 23), (LARL) 5 NHCH 3 (LARL4 6 ; SEQ ID NO: 24), KLLKLLLKLWLLKLLL (Hel-11-7; SEQ ID NO: 25), (KKKK) 2 GGC (KK; SEQ ID NO: 26), (KWKK) 2 GCC (KWK
• GRKKRRQRRRPPQ (SEQ ID NO: 38); the Drosophila melanogaster Antennapedia domain Antp (amino acids 43-58), RQIKIWFQNRRMKWKK (SEQ ID NO: 39); Buforin II,
• TRSSRAGLQFPVGRVHRLLRK (SEQ ID NO: 40) ; hClock-(amino acids 35-47) (human Clock protein DNA-binding peptide), KRVSRNKSEKKRR (SEQ ID NO: 41); MAP (model amphipathic peptide), KLALKLALKALKAALKLA (SEQ ID NO: 42); K-FGF, AAVALLPAVLLALLAP (SEQ ID NO: 43); Ku70- derived peptide, comprising a peptide selected from the group comprising VPMLKE (SEQ ID NO: 44), VPMLK (SEQ ID NO: 45), PMLKE (SEQ ID NO: 46) or PMLK (SEQ ID NO: 47) ; Prion, Mouse Prpe (amino acids 1-28), MANLGYWLLALFVTMWTDVGLCKKRPKP (SEQ ID NO: 48); pVEC,
• LLIILRRRIRKQAHAHSK (SEQ ID NO: 49); Pep-I, KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 50); SynBI, RGGRLSYSRRRFSTSTGR (SEQ ID NO: 51); Transportan, G WTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 52); Transportan-10, AGYLLGKINLKALAALAKKIL (SEQ ID NO: 53); CADY, Ac-
• GLWRALWRLLRSLWRLLWRA-cysteamide (SEQ ID NO: 54); Pep-7, SDLWEMMMVSLACQY (SEQ ID NO: 55); HN-1, TSPLNIHNGQKL (SEQ ID NO: 56) ; VT5, DPKGDPKGVTVTVTVTVTGKGDPKPD (SEQ ID NO: 57) ; or pISL, RVIRVWFQNKRCKDKK (SEQ ID NO: 58) .
• the membrane permeating moiety is a lipid moiety, such as a C 10 -C 20 fatty acyl group, especially octadecanoyl (stearoyl; C 18 ), hexadecanoyl (palmitoyl; C 16 ) or tetradecanoyl (myristoyl; C 14 ); most especially tetradecanoyl.
• the membrane permeable moiety is conjugated (attached) to the N- or C-terminal amino acid residue or through the amine of a lysine side-chain of the proteinaceous molecule, especially the N- terminal amino acid residue of the proteinaceous moiety.
• proteinaceous molecules with high levels of stability may be desired, for example, to increase the half-life of the proteinaceous molecule in a subject.
• the proteinaceous molecules of the invention comprise a stabilizing moiety, which is also referred to herein as a "protecting moiety" .
• stabilizing moiety may be conjugated at any point on the proteinaceous molecule.
• Suitable stabilizing moieties include polyethylene glycol (PEG) or a capping moiety, including an acetyl group, pyroglutamate or an amino group.
• the acetyl group and/or pyroglutamate are conjugated to the N-terminal amino acid residue of the proteinaceous molecule.
• the N-terminus of the proteinaceous molecule is a pyroglutamide or acetamide.
• the amino group is conjugated to the C-terminal amino acid residue of the proteinaceous molecule.
• the proteinaceous molecule of the invention has a primary amide at the C-terminus.
• the PEG is conjugated to the N-terminal or C-terminal amino acid residue of the proteinaceous molecule or through the amine of a lysine side-chain, especially through the N-terminal amino acid residue or through the amine of a lysine side-chain.
• the proteinaceous molecules of the invention have a primary amide or a free carboxyl group (C-terminal acid) at the C-terminus and a primary amine at the N- terminus.
• the proteinaceous molecules of the present invention are cyclic peptides. Without wishing to be bound by theory, cyclization of peptides is thought to decrease the susceptibility of the peptides to degradation.
• the proteinaceous molecules are cyclized using N-to-C cyclization (head to tail cyclization), preferably through an amide bond. Such peptides do not possess N- or C-terminal amino acid residues.
• the proteinaceous molecules of the invention have an amide-cyclized peptide backbone.
• the proteinaceous molecules of the invention are cyclized using side-chain to side-chain cyclization, preferably through a disulfide bond or a lactam bridge.
• the N- and C-termini are linked using a linking moiety.
• the linking moiety may be a peptide linker such that cyclization produces an amide-cyclized peptide backbone. Variation within the peptide sequence of the linking moiety is possible, such that the linking moiety may be modified to alter the physicochemical properties of the proteinaceous molecules and potentially reduce side effects of the proteinaceous molecules of the invention or otherwise improve the therapeutic use of the proteinaceous molecules, for example, by improving stability.
• the linking moiety will be of suitable length to span the distance between the N- and C- termini of the peptide without substantially altering the structural conformation of the
• a peptidic linking moiety may be between 2 and 10 amino acid residues in length. In some embodiments, longer or shorter peptidic linking moieties may be required.
• the proteinaceous molecules of the invention may be in the form of salts or prodrugs.
• the salts of the proteinaceous molecules of the present invention are preferably pharmaceutically acceptable, but it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present invention.
• the proteinaceous molecules of the present invention may be in crystalline form and/or in the form of solvates, for example, hydrates. Solvation may be performed using methods known in the art.
• the proteinaceous molecules of the invention selectively inhibit LSD1 over at least one other LSD or another enzyme such as a MAO. In some embodiments, the proteinaceous molecules of the invention selectively inhibit LSDl over the other LSD subtypes and MAOs. In some embodiments, the proteinaceous molecules of the invention exhibit LSDl selectivity of greater than about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or greater than about 100- fold with respect to inhibition of another LSD or MAO. In other embodiments, selective molecules display at least 50-fold greater inhibition towards LSDl than another LSD or MAO. In further embodiments, selective molecules display at least 100-fold greater inhibition towards LSDl than towards another LSD or MAO.
• selective molecules display at least 500-fold greater inhibition towards LSDl than towards another LSD or MAO. In yet further embodiments, selective molecules display at least 100-fold greater inhibition towards LSDl than towards another LSD or MAO.
• the proteinaceous molecules of the invention are non-selective LSDl inhibitors.
• the present invention also contemplates nucleic acid molecules which encode a proteinaceous molecule of the invention.
• an isolated nucleic acid molecule comprising a polynucleotide sequence that encodes a proteinaceous molecule of the invention or is complementary to a polynucleotide sequence that encodes a proteinaceous molecule of the invention, such as the proteinaceous molecule of Formula I; SEQ ID NO : 1, 2 or 3; or variant proteinaceous molecule as described herein.
• the proteinaceous molecule encoded by the polynucleotide sequence is other than a proteinaceous molecule consisting of the amino acid sequence of SEQ ID NO : 4.
• the isolated nucleic acid molecules of the present invention may be DNA or RNA.
• the nucleic acid When the nucleic acid is in DNA form, it may be genomic DNA or cDNA.
• RNA forms of the nucleic acid molecules of the present invention are generally mRNA.
• nucleic acid molecules are typically isolated, in some embodiments the nucleic acid molecules may be integrated into, ligated to, or otherwise fused or associated with other genetic molecules, such as an expression vector.
• an expression vector includes transcriptional and translational regulatory nucleic acid operably linked to the polynucleotide sequence. Accordingly, in another aspect of the invention, there is provided an expression vector comprising a polynucleotide sequence that encodes a proteinaceous molecule of the invention, such as the proteinaceous molecule of Formula I; SEQ ID NO: 1, 2 or 3; or variant proteinaceous molecule as described herein.
• the proteinaceous molecules of the invention may be produced inside a cell by introduction of one or more expression constructs, such as an expression vector, that comprise a polynucleotide sequence that encodes a proteinaceous molecule of the invention.
• the invention contemplates recombinantly producing the proteinaceous molecules of the invention inside a host cell, such as a mammalian cell (e.g. Chinese hamster ovary (CHO) cell, mouse myeloma (NSO) cell, baby hamster kidney (BHK) cell or human embryonic kidney (HEK293) cell), yeast cell (e.g. Pichia pastoris cell, Saccharomyces cerevisiae cell, Schizosaccharomyces pombe cell, Hansenula polymorpha cell, Kluyveromyces lactis cell, Yarrowia lipolytica cell or Arxula adeninivorans cell), or bacterial cell (e.g. Escherichia coli cell, Corynebacterium glutamicum or Pseudomonas fluorescens cell).
• a mammalian cell e.g. Chinese hamster ovary (CHO) cell, mouse myeloma (NSO) cell, baby
• proteinaceous molecules of the invention in vivo inside an LSD1, PKC- ⁇ , PD-L1 and/or PD-L2 overexpressing cell, especially an LSD1 overexpressing cell, such as a vertebrate cell, particularly a mammalian or avian cell, especially a mammalian cell.
• the expression of natural or synthetic nucleic acids is typically achieved by operably linking a polynucleotide sequence encoding a proteinaceous molecule of the invention to a regulatory element (e.g. a promoter, which may be either constitutive or inducible), suitably incorporating the construct into an expression vector and introducing the vector into a suitable host cell.
• a regulatory element e.g. a promoter, which may be either constitutive or inducible
• Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences and promoters useful for regulation of the expression of the nucleic acid.
• the vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, prokaryotes or both, (e.g.
• Vectors may be suitable for replication and integration in prokaryotes, eukaryotes, or both. See, Giliman and Smith (1979), Gene, 8 : 81-97; Roberts et al. (1987) Nature, 328 : 731-734; Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, volume 152, Academic Press, Inc., San Diego, Calif. (Berger) ; Sambrook et al. (1989), Molecular Cloning - a Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y.
• Expression vectors containing regulatory elements from eukaryotic viruses are typically used for expression of nucleic acid sequences in eukaryotic cells.
• SV40 vectors include pSVT7 and pMT2.
• Vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205.
• exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAM neo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
• viral expression vectors are useful for modifying eukaryotic cells because of the high efficiency with which the viral vectors transfect target cells and integrate into the target cell genome.
• Illustrative expression vectors of this type can be derived from viral DNA sequences including, but not limited to, adenovirus, adeno- associated viruses, herpes-simplex viruses and retroviruses such as B, C, and D retroviruses as well as spumaviruses and modified lentiviruses.
• Suitable expression vectors for transfection of animal cells are described, for example, by Wu and Ataai (2000) Curr. Opin. Biotechnol.
• the polypeptide or peptide-encoding portion of the expression vector may comprise a naturally-occurring sequence or a variant thereof, which has been engineered using recombinant techniques.
• the codon composition of a polynucleotide encoding a proteinaceous molecule of the invention is modified to permit enhanced expression of the proteinaceous molecule of the invention in a mammalian host using methods that take advantage of codon usage bias, or codon translational efficiency in specific mammalian cell or tissue types as set forth, for example, in International Publications WO 99/02694 and WO 00/42215.
• codon-optimized polynucleotides at least one existing codon of a parent polynucleotide is replaced with a synonymous codon that has a higher translational efficiency in a target cell or tissue than the existing codon it replaces.
• the replacement step affects 5%, 10%, 15%, 20%, 25%, 30%, more preferably 35%, 40%, 50%, 60%, 70% or more of the existing codons of a parent polynucleotide.
• the expression vector is compatible with the cell in which it is introduced such that the proteinaceous molecule of the invention is expressible by the cell.
• the expression vector is introduced into the cell by any suitable means which will be dependent on the particular choice of expression vector and cell employed. Such means of introduction are well-known to those skilled in the art. For example, introduction can be effected by use of contacting (e.g. in the case of viral vectors), electroporation, transformation, transduction, conjugation or triparental mating, transfection, infection membrane fusion with cationic lipids, high-velocity bombardment with DNA- coated microprojectiles, incubation with calcium phosphate-DNA precipitate, direct microinjection into single cells, and the like.
• the vectors are introduced by means of cationic lipids, e.g., liposomes.
• liposomes are commercially available (e.g. Lipofectin®, LipofectamineTM, and the like, supplied by Invitrogen Waltham MA, USA).
• the proteinaceous molecules of the present invention may be prepared using recombinant DNA techniques or by chemical synthesis.
• the proteinaceous molecules of the present invention are prepared using standard peptide synthesis methods, such as solution synthesis or solid phase synthesis.
• the chemical synthesis of the proteinaceous molecules of the invention may be performed manually or using an automated synthesizer.
• the linear peptides may be synthesized using solid phase peptide synthesis using either Boc or Fmoc chemistry, as described in Merrifield (1963) J Am Chem Soc, 85(14) : 2149-2154; Schnolzer, et al. (1992) Int J Pept Protein Res, 40 : 180-193; Ensenat-Waser, et a/.
• the proteinaceous molecules of the invention may be cyclized.
• the proteinaceous molecules of the present invention are prepared using recombinant DNA techniques.
• the proteinaceous molecules of the invention may be prepared by a procedure including the steps of: (a) preparing a construct comprising a polynucleotide sequence that encodes the proteinaceous molecule of the invention and that is operably linked to a regulatory element; (b) introducing the construct into a host cell; (c) culturing the host cell to express the polynucleotide sequence to thereby produce the encoded proteinaceous molecule of the invention ; and (d) isolating the proteinaceous molecule of the invention from the host cell.
• the proteinaceous molecule of the present invention may be prepared recombinantly using standard protocols, for example, as described in Klint, et al. (2013) PLOS One, 8(5) : e63865; Sambrook, et al. (1989) Molecular Cloning : A Laboratory Manual (Cold Spring Harbour Press), in particular Sections 16 and 17; Ausubel, et al. (1998) Current Protocols in Molecular Biology (John Wiley and Sons, Inc.), in particular Chapters 10 and 16; and Coligan, et al. (1997) Current Protocols in Protein Science (John Wiley and Sons, Inc.), in particular Chapters 1, 5 and 6.
• the LSD1 inhibitors are useful in compositions and methods for the inhibition of immune checkpoints, particularly the inhibition of PD-L1 and/or PD-L2.
• the proteinaceous molecules of the invention are also useful in compositions and methods for the treatment or prevention of a condition involving LSD1, PKC, PD-L1 and/or PD-L2 overexpression, such as a cancer or infection.
• the LSD1 inhibitors may be in the form of a pharmaceutical composition, wherein the pharmaceutical composition comprises an LSD1 inhibitor and a pharmaceutically acceptable carrier or diluent.
• the LSD1 inhibitor is a proteinaceous molecule of the invention.
• the LSD1 inhibitor may be formulated into the pharmaceutical composition as a neutral or salt form.
• compositions of the invention may be administered through a variety of routes including, but not limited to, oral, rectal, topical, intranasal, intraocular, transmucosal, intestinal, enteral, intramuscular, subcutaneous,
• the pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions and sterile powders for the preparation of sterile injectable solutions. Such forms should be stable under the conditions of manufacture and storage and may be preserved against reduction, oxidation and microbial contamination.
• Buffer systems are routinely used to provide pH values of a desired range and may include, but are not limited to, carboxylic acid buffers, such as acetate, citrate, lactate, tartrate and succinate; glycine; histidine; phosphate; tris(hydroxymethyl)aminomethane (Tris) ; arginine; sodium hydroxide; glutamate; and carbonate buffers.
• carboxylic acid buffers such as acetate, citrate, lactate, tartrate and succinate
• Tris tris(hydroxymethyl)aminomethane
• Suitable antioxidants may include, but are not limited to, phenolic compounds such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole; vitamin E; ascorbic acid ; reducing agents such as methionine or sulphite; metal chelators such as ethylene diamine tetraacetic acid (EDTA) ; cysteine hydrochloride; sodium bisulfite; sodium metabisulfite; sodium sulfite; ascorbyl palmitate; lecithin ; propyl gallate; and alpha-tocopherol.
• BHT butylated hydroxytoluene
• reducing agents such as methionine or sulphite
• metal chelators such as ethylene diamine tetraacetic acid (EDTA)
• cysteine hydrochloride sodium bisulfite; sodium metabisulfite; sodium sulfite; ascorbyl palmitate; lecithin ; propyl gallate; and alpha
• the LSD1 inhibitors may be formulated in aqueous solutions, suitably in physiologically compatible buffers such as Hanks' solution, Ringer's solution or physiological saline buffer.
• physiologically compatible buffers such as Hanks' solution, Ringer's solution or physiological saline buffer.
• penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
• compositions of the present invention may be formulated for administration in the form of liquids, containing acceptable diluents (such as saline and sterile water), or may be in the form of lotions, creams or gels containing acceptable diluents or carriers to impart the desired texture, consistency, viscosity and appearance.
• acceptable diluents such as saline and sterile water
• Acceptable diluents and carriers are familiar to those skilled in the art and include, but are not restricted to, ethoxylated and nonethoxylated surfactants, fatty alcohols, fatty acids, hydrocarbon oils (such as palm oil, coconut oil, and mineral oil), cocoa butter waxes, silicon oils, pH balancers, cellulose derivatives, emulsifying agents such as non-ionic organic and inorganic bases, preserving agents, wax esters, steroid alcohols, triglyceride esters, phospholipids such as lecithin and cephalin, polyhydric alcohol esters, fatty alcohol esters, hydrophilic lanolin derivatives and hydrophilic beeswax derivatives.
• ethoxylated and nonethoxylated surfactants include, but are not restricted to, ethoxylated and nonethoxylated surfactants, fatty alcohols, fatty acids, hydrocarbon oils (such as palm oil, coconut oil, and mineral oil), cocoa butter waxes, silicon oils
• the LSD1 inhibitors can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration, which is also contemplated for the practice of the present invention.
• Such carriers enable the bioactive agents of the invention to be formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
• These carriers may be selected from sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and pyrogen-free water.
• compositions for parenteral administration include aqueous solutions of the LSD1 inhibitors in water-soluble form. Additionally, suspensions of the LSD1 inhibitors may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
• Sterile solutions may be prepared by combining the active compounds in the required amount in the appropriate solvent with other excipients as described above as required, followed by sterilization, such as filtration.
• dispersions are prepared by incorporating the various sterilized active compounds into a sterile vehicle which contains the basic dispersion medium and the required excipients as described above.
• Sterile dry powders may be prepared by vacuum- or freeze-drying a sterile solution comprising the active compounds and other required excipients as described above.
• compositions for oral use can be obtained by combining the LSD1 inhibitors with solid excipients and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
• suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatine, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or
• compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more therapeutic agents as described above with the carrier which constitutes one or more necessary ingredients.
• the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g. by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• Dragee cores are provided with suitable coatings.
• suitable coatings may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of particle doses.
• compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added.
• LSD1 inhibitors may be incorporated into modified-release preparations and formulations, for example, polymeric microsphere formulations, and oil- or gel-based formulations.
• the LSD1 inhibitors may be administered in a local rather than systemic manner, such as by injection of the LSD1 inhibitor directly into a tissue, which is preferably subcutaneous or omental tissue, often in a depot or sustained release formulation.
• the LSD1 inhibitors are systemically administered.
• the LSD1 inhibitors may be administered in a targeted drug delivery system, such as in a particle which is suitable targeted to and taken up selectively by a cell or tissue.
• the LSD1 inhibitor is contained or otherwise associated with a vehicle selected from liposomes, micelles, dendrimers, biodegradable particles, artificial DNA nanostructure, lipid-based nanoparticles and carbon or old nanoparticles.
• the vehicle is selected from poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) (PEG), PLA-PEG copolymers and combinations thereof.
• the effective local concentration of the agent may not be related to plasma concentration.
• compositions in dosage unit form for ease of administration and uniformity of dosage.
• determination of the novel dosage unit forms of the present invention is dictated by and directly dependent on the unique characteristics of the active material, the particular therapeutic effect to be achieved and the limitations inherent in the art of compounding active materials for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
• the LSD1 inhibitor may be the sole active ingredient administered to the subject, the administration of other active ingredients concurrently with said LSD1 inhibitor is within the scope of the invention.
• the LSD1 inhibitor may be administered concurrently with one or more cancer therapies or anti-infective agents.
• the LSD1 inhibitor may be therapeutically used after the cancer therapy or anti-infective agent or may be therapeutically used together with the cancer therapy or anti-infective agent.
• the LSD1 inhibitor may be administered separately, simultaneously or sequentially with the other active ingredient.
• composition comprising an LSD1 inhibitor and an anti-infective agent.
• present invention also contemplates a composition comprising an LSD1 inhibitor and a cancer therapy.
• Suitable cancer therapies include, but are not limited to, radiotherapy, surgery, chemotherapy, hormone ablation therapy, pro-apoptosis therapy and immunotherapy.
• Suitable radiotherapies include radiation and waves that induce DNA damage, for example, ⁇ -irradiation, X-rays, UV irradiation, microwaves, electronic emissions and radioisotopes.
• therapy may be achieved by irradiating the localized tumor site with the above described forms of radiations. It is most likely that all of these factors cause a broad range of damage to DNA, on the precursors of DNA, on the replication and repair of DNA and on the assembly and maintenance of chromosomes.
• the dosage range for X-rays ranges from daily doses of 50-200 roentgens for prolonged periods of time such as 3-4 weeks, to single doses of 2000-6000 roentgens.
• Dosage ranges for radioisotopes vary widely and depend on the half life of the isotope, the strength and type of radiation emitted and the uptake by the neoplastic cells.
• Suitable radiotherapies may include, but are not limited to, conformal external beam radiotherapy (50-100 Gray given as fractions over 4-8 weeks), either single shot or fractionated high dose brachytherapy, permanent interstitial brachytherapy and systemic radioisotopes such as Strontium 89.
• the radiotherapy may be administered with a radiosensitizing agent.
• Suitable radiosensitizing agents may include, but are not limited to, efaproxiral, etanidazole, fluosol, misonidazole, nimorazole, temoporfin and tirapazamine.
• Suitable chemotherapeutic agents may include, but are not limited to, antiproliferative/antineoplastic drugs and combinations thereof including alkylating agents (for example cisplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan and nitrosoureas), antimetabolites (for example antifolates such as fluoropyridines like 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside and hydroxyurea), antitumor antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin), antimitotic agents (for example Vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and tax
• inhibitors of growth factor function include growth factor antibodies, growth factor receptor antibodies (for example the anti-erbb2 antibody trastuzumab [HerceptinTM] and the anti-erbbl antibody Cetuximab [C225]), farnesyl transferase inhibitors, M EK inhibitors, tyrosine kinase inhibitors and serine/threonine kinase inhibitors, for example other inhibitors of the epidermal growth factor family (for example other EGFR family tyrosine kinase inhibitors such as N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3- morpholinopropoxy)quinazolin-4-amine (Gefitinib, AZD1839), N-(3-ethynylphenyl)-6,7-bis
• Suitable immunotherapy approaches may include, but are not limited to ex vivo and in vivo approaches to increase the immunogenicity of patient tumor cells such as transfection with cytokines including interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor; approaches to decrease T-cell anergy; approaches using transfected immune cells such as cytokine-transfected dendritic cells; approaches using cytokine-transfected tumor cell lines; and approaches using anti-idiotypic antibodies.
• cytokines including interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor
• approaches to decrease T-cell anergy approaches using transfected immune cells such as cytokine-transfected dendritic cells
• approaches using cytokine-transfected tumor cell lines and approaches using anti-idiotypic antibodies.
• the immune effector may be, for example, an antibody specific for some marker on the surface of a malignant cell.
• the antibody alone may serve as an effector of therapy or it may recruit other cells to actually facilitate cell killing.
• the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
• the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a malignant cell target.
• Various effector cells include cytotoxic T cells and NK cells.
• Examples of other cancer therapies include phytotherapy, cryotherapy, toxin therapy or pro-apoptosis therapy.
• phytotherapy phytotherapy
• cryotherapy toxin therapy
• pro-apoptosis therapy pro-apoptosis therapy
• Suitable anti-infective agents include, but are not limited to, antimicrobials, which may include, but are not limited to, compounds that kill or inhibit the growth of microorganisms such as viruses, bacteria, yeast, fungi, protozoa, etc. and, thus, include antibiotics, amebicides, antifungals, antiprotozoals, antimalarials, antituberculotics and antivirals.
• Anti-infective drugs also include within their scope anthelmintics and nematocides.
• antibiotics include quinolones (e.g. amifloxacin, cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin, oxolinic acid, pefloxacin, rosoxacin, temafloxacin, tosufloxacin, sparfloxacin, clinafloxacin, gatifloxacin, moxifloxacin, gemifloxacin, trovafloxacin and garenoxacin), tetracyclines,
• quinolones e.g. amifloxacin, cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin,
• glycylcyclines and oxazolidinones e.g. chlortetracycline, demeclocycline, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, tetracycline, tigecycline, linezolid, tedizolid and eperezolid
• glycopeptides e.g. aminoglycosides
• amikacin arbekacin, butirosin, dibekacin, fortimicins, gentamicin, kanamycin, menomycin, neomycin, netilmicin, paromomycin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin), ⁇ -lactams (e.g.
• ketolides e.g. telithromycin, cethromycin
• coumermycins e.g. clindamycin, lincomycin
• chloramphenicol and salts and combinations thereof.
• Illustrative antivirals include abacavir, acyclovir, adefovir, amantadine, amprenavir, atazanavir, boceprevir, cidofovir, daclatasvir, darunavir, delavirdine, didanosine, dolutegravir, efavirenz, elvitegravir, emtricitabine, enfuvirtide, entacavir, etravirine, famciclovir, fomivirsen, fosamprenavir, foscarnet, ganciclovir, indinavir, lamivudine, lamivudine/zidovudine, lopenavir, maraviroc, nelfinavir, nevirapine, oseltamivir, PEG-interferon alpha-2b, peramivir, raltegravir, ribavirin, rilpivirine
• Suitable amebicides or antiprotozoals include, but are not limited to, atovaquone, chloroquine, iodoquinol, mefloquine, metronidazole, nitazoxanide, paramomycin, pentamidine, tinidazole and salts and combinations thereof.
• Anthelmintics can be at least one selected from mebendazole, pyrantel, praziquantel, miltefosine, albendazole, ivermectin, thiabendazole and salts and combinations thereof.
• Illustrative antifungals can be selected from amphotericin B, amphotericin B cholesteryl sulfate complex, amphotericin B lipid complex, amphotericin B liposomal, anidulafungin, caspofungin, clotrimazole, fluconazole, flucytosine, griseofulvin, griseofulvin microsize, griseofulvin ultramicrosize, isavuconazonium, itraconazole, ketoconazole, micafungin, miconazole, nystatin, posaconazole, terbinafine, voriconazole and salts and combinations thereof.
• Suitable antimalarials include, but are not limited to, chloroquine, doxycycline, hydroxychloroquine, mefloquine, primaquine, pyrimethamine, pyrimethamine with sulfadoxine, quinine and salts and combinations thereof.
• Antituberculotics include but are not restricted to aminosalicylic acid, bedaquiline, capreomycin, clofazimine, cycloserine, dapsone, ethambutol, ethionamide, isoniazid, pyrazinamide, rifabutin, rifampin, rifapentine, streptomycin and salts and combinations thereof.
• the LSD1 inhibitor may be compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form.
• a unit dosage form may comprise the LSD1 inhibitor in an amount in the range of from about 0.25 pg to about 2000 mg.
• the LSD1 inhibitor may be present in an amount of from about 0.25 pg to about 2000 mg/mL of carrier.
• the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
• LSD1 inhibitors inhibit immune checkpoints, particularly PD-L1 and/or PD-L2, including the nuclear translocation of PD-L1 and/or PD-L2, especially PD-L1. Accordingly, the inventors have conceived that LSD1 inhibitors may be used for a range of applications, including for enhancing an immune response in a subject to a target antigen by an immune-modulating agent, for enhancing the efficacy of an anti-infective or for the treatment of a cancer such as a metastatic cancer or an infection.
• a method of inhibiting PD-L1 and/or PD-L2 activity comprising contacting a PD-L1 and/or PD-L2 overexpressing cell with an LSDl inhibitor.
• the present invention also contemplates an LSDl inhibitor for use in inhibiting PD- Ll and/or PD-L2 activity.
• the inhibition of PD-L1 and/or PD-L2 activity includes, but is not limited to, the inhibition of the interaction of PD-L1 and/or PD-L2 with PD-1, the expression of PD-L1 and/or PD- L2, or the nuclear translocation of PD-L1 and/or PD-L2.
• a method of inhibiting the nuclear translocation of PD-L1 and/or PD-L2 in a PD-L1 and/or PD-L2 overexpressing cell comprising contacting the PD-L1 and/or PD-L2 overexpressing cell with an LSDl inhibitor.
• the present invention also provides a use of an LSDl inhibitor for inhibiting the nuclear translocation of PD-L1 and/or PD-L2 in a PD-L1 and/or PD-L2 overexpressing cell; an LSDl inhibitor for use in inhibiting the nuclear translocation of PD-L1 and/or PD-L2 in a PD-L1 and/or PD-L2 overexpressing cell; and the use of an LSDl inhibitor in the manufacture of a medicament for inhibiting the nuclear translocation of PD-L1 and/or PD-L2 in a PD-L1 and/or PD-L2 overexpressing cell.
• the PD-L1 and/or PD-L2 overexpressing cell is a cancer stem cell or a non-cancer stem cell tumor cell, especially a cancer stem cell tumor cell.
• the present invention also contemplates an LSDl inhibitor for use in inhibiting PD-L1 and/or PD-L2 activity in a subject, and the use of an LSDl inhibitor in the manufacture of a medicament for inhibiting PD-L1 and/or PD-L2 activity in a subject.
• the subject has elevated PD-L1 and/or PD-L2 activity.
• the LSDl inhibitor inhibits the nuclear translocation of PD-L1 and/or PD-L2 in a subject, especially PD-L1.
• the present invention also extends to a method of inhibiting the nuclear translocation of PD-L1 and/or PD-L2 in subject, comprising administering an LSDl inhibitor to the subject.
• the invention provides a use of an LSDl inhibitor for inhibiting the nuclear translocation of PD-L1 and/or PD-L2 in a subject.
• the invention also provides a use of an LSDl inhibitor in the manufacture of a medicament for inhibiting the nuclear translocation of PD-L1 and/or PD-L2 in a subject, and an LSDl inhibitor for use in inhibiting the nuclear translocation of PD-L1 and/or PD-L2 in a subject.
• the LSDl is a selective LSDl inhibitor. In alternative embodiments, the LSDl inhibitor is a non-selective LSDl inhibitor.
• the present invention also contemplates the use of an LSDl inhibitor for the treatment of a condition in which inhibition of PD-L1 and/or PD-L2 activity is associated with effective treatment, including increased efficacy of treatment.
• the present invention also provides a method for treating a condition in a subject in which inhibition of PD-L1 and/or PD-L2 is associated with effective treatment, comprising administering an LSDl inhibitor to the subject.
• the present invention provides the use of an LSDl inhibitor in the manufacture of a medicament for treating a condition in a subject in which inhibition of PD-L1 and/or PD-L2 is associated with effective treatment.
• Conditions involving PD-L1 and/or PD-L2 activity include, but are not limited to, a cancer particularly a metastatic cancer, or an infection, particularly a pathogenic infection.
• the subject has a cancer or an infection.
• the subject has a cancer, particularly a metastatic cancer.
• the cancer is selected from, but is not limited to, breast, prostate, lung, bladder, pancreatic, colon, melanoma, retinoblastoma, liver or brain cancer;
• breast cancer especially breast cancer; most especially metastatic breast cancer.
• the subject has an infection, particularly a pathogenic infection.
• the infection may be selected from, but is not limited to, a viral, bacterial, yeast, fungal, helminth or protozoan infection.
• Viral infections contemplated by the present invention include, but are not restricted to, infections caused by HIV, hepatitis, influenza virus, Japanese encephalitis virus, Epstein-Barr virus, herpes simplex virus, filovirus, human papillomavirus, human T-cell lymphotropic virus, human retrovirus, cytomegalovirus, varicella-zoster virus, poliovirus, measles virus, rubella virus, mumps virus, adenovirus, enterovirus, rhinovirus, ebola virus, west nile virus and respiratory syncytial virus; especially infections caused by HIV, hepatitis, influenza virus, Japanese encephalitis virus, Epstein-Barr virus and respiratory syncytial virus.
• Bacterial infections include, but are not restricted to, those caused by Neisseria species, Meningococcal species, Haemophilus species, Salmonella species, Streptococcal species, Legionella species, Mycoplasma species, Bacillus species, Staphylococcus species, Chlamydia species, Actinomyces species, Anabaena species, Bacteroides species, Bdellovibrio species, Bordetella species, Borrelia species, Campylobacter species, Caulobacter species, Chlrorbium species, Chromatium species,
• Hyphomicrobium species Leptospira species, Listeria species, Micrococcus species, Myxococcus species, Nitrobacter species, Oscillatoria species, Prochloron species, Proteus species,
• Rhodospirillum species Rhodospirillum species, Rickettsia species, Shigella species, Spirillum species, Spirochaeta species, Streptomyces species, Thiobacillus species, Treponema species, Vibrio species, Yersinia species, Nocardia species and Mycobacterium species; especially infections caused by Neisseria species, Meningococcal species, Haemophilus species, Salmonella species, Streptococcal species, Legionella species and Mycobacterium species.
• Protozoan infections encompassed by the invention include, but are not restricted to, those caused by Plasmodium species, Leishmania species, Trypanosoma species, Toxoplasma species, Entamoeba species and Giardia species.
• Helminth infections may include, but are not limited to, infections caused by
• Fungal infections contemplated by the present invention include, but are not limited to, infections caused by Histoplasma species and Candida species.
• the LSDl inhibitor is a proteinaceous molecule comprising, consisting or consisting essentially of a sequence corresponding to residues 108 to 118 of LSDl as broadly described above; particularly a proteinaceous molecule comprising, consisting or consisting essentially of Formula I, SEQ ID NO: 1, 2 or 3, or a variant thereof.
• the present invention also extends to the use of an LSD1 inhibitor for enhancing the efficacy of an anti-infective agent. Accordingly, in another aspect of the invention, there is provided a method of enhancing the efficacy of an anti-infective agent in a subject, comprising administering an LSD1 inhibitor to the subject.
• the invention also provides the use of an LSD1 inhibitor in the manufacture of a medicament for enhancing the efficacy of an anti-infective agent in a subject, and an LSD1 inhibitor for use in enhancing the efficacy of an anti-infective agent in a subject.
• the anti-infective agent may include, but is not limited to, an antimicrobial, which may include, but is not limited to, any compound that kills or inhibit the growth of microorganisms such as viruses, bacteria, yeast, fungi, protozoa, etc. and, thus, includes antibiotics, amebicides, antifungals, antiprotozoals, antimalarials, antituberculotics and antivirals.
• Anti-infective agents also include within their scope anthelmintics and nematocides. Illustrative antibiotics, amebicides, antifungals, antiprotozoals, antimalarials, antituberculotics and antivirals are described in Section 3 supra.
• the subject has an infection, especially a pathogenic infection.
• the infection may be selected from, but is not limited to, a viral, bacterial, yeast, fungal, helminth or protozoan infection as described supra.
• an LSD1 inhibitor is useful for enhancing an immune response in a subject to a target antigen by an immune-modulating agent.
• the present invention also contemplates an LSD1 inhibitor for use in enhancing an immune response in a subject to a target antigen by an immune-modulating agent and the use of an LSD1 inhibitor in the manufacture of a medicament for enhancing an immune response in a subject to a target antigen by an immune-modulating agent.
• the LSD1 inhibitor and the immune-modulating agent may be administered
• compositions comprising an LSD1 inhibitor and an immune-modulating agent.
• Such compositions can be administered to a subject either by themselves or in formulations where they are combined with a pharmaceutically acceptable carrier or diluent. Suitable formulations are described in Section 3.
• the immune-modulating agent includes, but is not limited to, an antigen that corresponds to at least a portion of the target antigen, an antigen binding molecule that is immuno-interactive with the target antigen and an immune modulating cell that modulates an immune response to the target antigen.
• the antigen may be any antigen that corresponds to at least a portion of a target antigen of interest for stimulating an immune response to the target antigen.
• a target antigen of interest for stimulating an immune response to the target antigen.
• Such an antigen may be in soluble form (e.g. peptide, polypeptide or a nucleic acid molecule from which a peptide or polypeptide is expressible) or in the form of whole cells or attenuated pathogen preparations (e.g. attenuated virus or bacteria) or it may be presented by antigen-presenting cells as described in more detail below.
• Target antigens useful in the present invention can be any type of biological molecule including, for example, simple intermediary metabolites, sugars, lipids, and hormones as well as macromolecules such as complex carbohydrates, phospholipids, nucleic acids, polypeptides and peptides.
• Target antigens may be selected from endogenous antigens produced by a host or exogenous antigens that are foreign to the host. Suitable endogenous antigens may include, but are not limited to, cancer or tumor antigens.
• Non-limiting examples of cancer or tumor antigens include antigens from a cancer or tumor selected from ABL1 protooncogene, AIDS related cancers, acoustic neuroma, acute lymphocytic leukaemia, acute myeloid leukaemia, adenocystic carcinoma, adrenocortical cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft-part sarcoma, anal cancer, angiosarcoma, aplastic anaemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancers, bowel cancer, brain stem glioma, brain and CNS tumors, breast cancer, CNS tumors, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancer, childhood leukaemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leuk
• tumor-specific antigens include, but are not limited to : etv6, amll, cyclophilin b (acute lymphoblastic leukemia) ; Ig-idiotype (B cell lymphoma) ; E-cadherin, a-catenin, ⁇ -catenin, ⁇ -catenin, pl20ctn (glioma) ; p21ras (bladder cancer) ; p21ras (biliary cancer) ; M UC family, HER2/neu, c-erbB-2 (breast cancer) ; p53, p21ras (cervical carcinoma) ; p21ras, HER2/neu, c-erbB-2, M UC family, Cripto-lprotein, Pim-1 protein (colon carcinoma) ; Colorectal associated antigen (CRC)-C017-1A/GA733, APC (colorectal cancer) ; carcinoembry
• melanoma-specific antigens e.g. BAGE, GAGE-1, gpl00In4, MAGE-1 (e.g. GenBank Accession No. X54156 and AA494311), MAGE-3, MAGE4, PRAM E, TRP2IN2, NYNSOla, NYNSOlb, LAGE1, p97 melanoma antigen (e.g. GenBank Accession No.
• M 12154 p5 protein, gp75, oncofetal antigen, GM2 and GD2 gangliosides, cdc27, p21ras, gpl00 Pmel117 (melanoma) ; M UC family, p21ras (myeloma) ; HER2/neu, c-erbB-2 (non-small cell lung carcinoma) ; Imp-1, EBNA-1 (nasopharyngeal cancer) ; MUC family, HER2/neu, c-erbB-2, MAGE-A4, NY-ESO-1 (ovarian cancer) ; Prostate Specific Antigen (PSA) and its antigenic epitopes PSA-1, PSA-2, and PSA-3, PSMA, HER2/neu, c-erbB-2, ga733 glycoprotein (prostate cancer) ; HER2/neu, c-erbB-2 (renal cancer) ; viral products such as human papilloma virus proteins (squa
• Foreign antigens are suitably antigens from pathogenic organisms.
• Exemplary pathogenic organisms include, but are not limited to, viruses, bacteria, fungi parasites, algae and protozoa and amoebae.
• viruses include viruses responsible for diseases including, but not limited to, measles, mumps, rubella, poliomyelitis, hepatitis A, B (e.g. GenBank Accession No. E02707), and C (e.g. GenBank Accession No. E06890), as well as other hepatitis viruses, influenza, adenovirus (e.g. types 4 and 7), rabies (e.g. GenBank Accession No.
• Epstein-Barr virus and other herpesviruses such as papillomavirus, Ebola virus, influenza virus, Japanese encephalitis (e.g. GenBank Accession No. E07883), dengue (e.g. GenBank Accession No. M24444), hantavirus, Sendai virus, respiratory syncytial virus, othromyxoviruses, vesicular stomatitis virus, visna virus, cytomegalovirus and human immunodeficiency virus (HIV) (e.g. GenBank Accession No. U 18552). Any suitable antigen derived from such viruses are useful in the practice of the present invention.
• herpesviruses such as papillomavirus, Ebola virus, influenza virus, Japanese encephalitis (e.g. GenBank Accession No. E07883), dengue (e.g. GenBank Accession No. M24444), hantavirus, Sendai virus, respiratory syncytial virus,
• illustrative retroviral antigens derived from HIV include, but are not limited to, antigens such as gene products of the gag, pol, and env genes, the Nef protein, reverse transcriptase, and other HIV components.
• hepatitis viral antigens include, but are not limited to, antigens such as the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, and other hepatitis, e.g. hepatitis A, B, and C, viral components such as hepatitis C viral RNA.
• influenza viral antigens include, but are not limited to, antigens such as hemagglutinin and neuraminidase and other influenza viral components.
• measles viral antigens include, but are not limited to, antigens such as the measles virus fusion protein and other measles virus components.
• rubella viral antigens include, but are not limited to, antigens such as proteins El and E2 and other rubella virus components; rotaviral antigens such as VP7sc and other rotaviral components.
• cytomegaloviral antigens include, but are not limited to, antigens such as envelope glycoprotein B and other cytomegaloviral antigen components.
• respiratory syncytial viral antigens include antigens such as the RSV fusion protein, the M2 protein and other respiratory syncytial viral antigen components.
• herpes simplex viral antigens include, but are not limited to, antigens such as immediate early proteins, glycoprotein D, and other herpes simplex viral antigen components.
• varicella zoster viral antigens include antigens such as 9PI, gpll, and other varicella zoster viral antigen components.
• Non-limiting examples of Japanese encephalitis viral antigens include antigens such as proteins E, M-E, M-E-NS 1, NS 1, NS 1-NS2A, 80%E, and other Japanese encephalitis viral antigen components.
• rabies viral antigens include, but are not limited to, antigens such as rabies glycoprotein, rabies nucleoprotein and other rabies viral antigen components.
• papillomavirus antigens include, but are not limited to, the LI and L2 capsid proteins as well as the E6/E7 antigens associated with cervical cancers, refer to : Fundamental Virology, Second Edition, eds. Fields, B.N. and Knipe, D. M., 1991, Raven Press, New York, for additional examples of viral antigens.
• fungi include Acremonium spp., Aspergillus spp., Basidiobolus spp., Bipolaris spp., Blastomyces dermatidis, Candida spp., Cladophialophora carrionii,
• Coccoidiodes immitis Conidiobolus spp., Cryptococcus spp., Curvularia spp., Epidermophyton spp., Exophiala jeanselmei, Exserohilum spp., Fonsecaea compacta, Fonsecaea pedrosoi, Fusarium oxysporum, Fusarium solani, Geotrichum candidum, Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var.
• duboisii Hortaea wasneckii, Lacazia loboi, Lasiodiplodia theobromae, Leptosphaeria senegalensis, Madurella grisea, Madurella mycetomatis, Malassezia furfur,
• illustrative fungal antigens that can be used in the compositions and methods of the present invention include, but are not limited to, Candida fungal antigen components; histoplasma fungal antigens such as heat shock protein 60 (HSP60) and other histoplasma fungal antigen components; cryptococcal fungal antigens such as capsular polysaccharides and other cryptococcal fungal antigen components; coccidiodes fungal antigens such as spherule antigens and other coccidiodes fungal antigen components; and tinea fungal antigens such as trichophytin and other coccidiodes fungal antigen components.
• Candida fungal antigen components histoplasma fungal antigens such as heat shock protein 60 (HSP60) and other histoplasma fungal antigen components
• cryptococcal fungal antigens such as capsular polysaccharides and other cryptococcal fungal antigen components
• coccidiodes fungal antigens such as spherule anti
• bacteria include bacteria that are responsible for diseases including, but not restricted to, diphtheria (e.g. Corynebacterium diphtheria), pertussis (e.g.
• Bordetella pertussis GenBank Accession No. M35274
• tetanus e.g. Clostridium tetani, GenBank Accession No. M64353
• tuberculosis e.g. Mycobacterium tuberculosis
• bacterial pneumonias e.g. Haemophilus influenzae
• cholera e.g. Vibrio cholerae
• anthrax e.g. Bacillus anthracis
• typhoid plague, shigellosis (e.g. Shigella dysenteriae)
• botulism e.g. Clostridium botulinum
• salmonellosis e.g. GenBank Accession No.
• peptic ulcers e.g. Helicobacter pylori
• Legionnaire's Disease Lyme disease
• Other pathogenic bacteria include Escherichia coli, Clostridium perfringens, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes.
• bacterial antigens which can be used in the compositions and methods of the invention include, but are not limited to, pertussis bacterial antigens such as pertussis toxin, filamentous hemagglutinin, pertactin, F M2, FIM3, adenylate cyclase and other pertussis bacterial antigen components; diphtheria bacterial antigens such as diphtheria toxin or toxoid and other diphtheria bacterial antigen components; tetanus bacterial antigens such as tetanus toxin or toxoid and other tetanus bacterial antigen components; streptococcal bacterial antigens such as M proteins and other streptococcal bacterial antigen components; gram-negative bacilli bacterial antigens such as lipopolysaccharides and other gram-negative bacterial antigen components; Mycobacterium tuberculosis bacterial antigens such as mycolic acid, heat
• protozoa examples include protozoa that are responsible for diseases including, but not limited to, malaria (e.g. GenBank Accession No. X53832), hookworm, onchocerciasis (e.g. GenBank Accession No. M27807), schistosomiasis (e.g. GenBank Accession No. LOS 198), toxoplasmosis, trypanosomiasis, leishmaniasis, giardiasis (GenBank Accession No. M33641), amoebiasis, filariasis (e.g. GenBank Accession No. J03266), borreliosis and trichinosis.
• malaria e.g. GenBank Accession No. X53832
• hookworm e.g. GenBank Accession No. M27807
• schistosomiasis e.g. GenBank Accession No. LOS 198
• toxoplasmosis trypanosomiasis
• protozoal antigens which can be used in the compositions and methods of the invention include, but are not limited to, Plasmodium falciparum antigens such as merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte/gamete surface antigens, blood-stage antigen pf 155/RESA and other plasmodial antigen components; toxoplasma antigens such as SAG-1, p30 and other toxoplasmal antigen components; schistosomae antigens such as glutathione-S-transferase, paramyosin, and other schistosomal antigen components; leishmania major and other leishmaniae antigens such as gp63, lipophosphoglycan and its associated protein and other leishmanial antigen components; and Trypanosoma cruzi antigens such as the 75-77kDa antigen, the 56kDa antigen and other trypanosomal
• the present invention also contemplates toxin components as antigens.
• toxins include, but are not restricted to, staphylococcal enterotoxins, toxic shock syndrome toxin, retroviral antigens (e.g. antigens derived from HIV), streptococcal antigens, staphylococcal enterotoxin-A (SEA), staphylococcal enterotoxin-B (SEB), staphylococcal enterotoxini-3 (SE ⁇ ), staphylococcal enterotoxin-D (SED), staphylococcal enterotoxin-E (SEE) as well as toxins derived from mycoplasma, mycobacterium and herpes viruses.
• staphylococcal enterotoxins include, but are not restricted to, staphylococcal enterotoxins, toxic shock syndrome toxin, retroviral antigens (e.g. antigens derived from HIV), streptococcal antigens, staphylococcal enterotoxin-A (SEA), sta
• the antigen corresponding to at least a portion of the target antigen may be isolated from a natural source or may be prepared by recombinant techniques as known in the art.
• peptide antigens can be eluted from the MHC and other presenting molecules of antigen- presenting cells obtained from a cell population or tissue for which a modified immune response is desired.
• the eluted peptides can be purified using standard protein purification techniques known in the art (Rawson et al. (2000), Cancer Res, 60(16) : 4493-4498). If desired, the purified peptides can be sequenced and synthetic versions of the peptides produced using standard protein synthesis techniques as for example described below.
• crude antigen preparations can be produced by isolating a sample of a cell population or tissue for which a modified immune response is desired, and either lysing the sample or subjecting the sample to conditions that will lead to the formation of apoptotic cells (e.g. irradiation with ultra violet or with ⁇ rays, viral infection, cytokines or by depriving cells of nutrients in the cell culture medium, incubation with hydrogen peroxide, or with drugs such as dexamethasone, ceramide chemotherapeutics and anti-hormonal agents such as Lupron or Tamoxifen).
• the lysate or the apoptotic cells can then be used as a source of crude antigen for use in soluble form or for contact with antigen-presenting cells as described in more detail below.
• the antigen When the antigen is known, it may be conveniently prepared in recombinant form using standard protocols as for example described in : Sambrook et al. (1989) Molecular Cloning : A Laboratory manual (Cold Spring Harbor Press), in particular Sections 16 and 17; Ausubel et al. (1998), Current Protocols in Molecular Biology (John Wiley & Sons, Inc.), in particular Chapters 10 and 16; and Coligan et al. (1997), Current Protocols in Protein Science (John Wiley & Sons, Inc.), in particular Chapters 1, 5 and 6.
• an antigen may be prepared by a procedure including the steps of (a) providing an expression vector from which the target antigen or analogue or mimetic thereof is expressible; (b) introducing the vector into a suitable host cell ; (c) culturing the host cell to express recombinant polypeptide from the vector; and (d) isolating the recombinant polypeptide.
• the expression vector will comprise an antigen-encoding polynucleotide which is operably connected to a regulatory polynucleotide.
• the antigen-encoding polynucleotide can be constructed from any suitable parent polynucleotide that codes for an antigen that corresponds to the target antigen of interest.
• the parent polynucleotide is suitably a natural gene or portion thereof. However, it is possible that the parent polynucleotide is not naturally-occurring but has been engineered using recombinant techniques.
• the regulatory polynucleotide suitably comprises transcriptional and/or translational control sequences, which will generally be appropriate for the host cell used for expression of the antigen-encoding polynucleotide.
• the transcriptional and translational regulatory control sequences include, but are not limited to, a promoter sequence, a 5' non-coding region, a c/ ' s-regulatory region such as a functional binding site for transcriptional regulatory protein or translational regulatory protein, an upstream open reading frame, transcriptional start site, translational start site, and/or nucleotide sequence which encodes a leader sequence, termination codon, translational stop site and a 3' non-translated region.
• Constitutive or inducible promoters as known in the art are contemplated by the invention.
• the promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.
• Promoter sequences contemplated by the present invention may be native to the host cell to be introduced or may be derived from an alternative source, where the region is functional in the host cell.
• the expression vector may also comprise a 3' non-translated sequence, which usually refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression.
• the polyadenylation signal is characterised by effecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. Polyadenylation signals are commonly recognised by the presence of homology to the canonical form 5' AATAAA-3' although variations are not uncommon.
• the 3' non- translated regulatory DNA sequence typically includes from about 50 to 1,000 nucleotide base pairs and may contain transcriptional and translational termination sequences in addition to a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression.
• the expression vector further contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.
• the expression vector may also include a fusion partner (typically provided by the expression vector) so that the recombinant polypeptide is expressed as a fusion polypeptide with the fusion partner.
• a fusion partner typically provided by the expression vector
• the main advantage of fusion partners is that they assist identification and/or purification of said fusion polypeptide.
• fusion partners include, but are not limited to, glutathione-S-transferase (GST), Fc portion of human IgG, maltose binding protein (M BP) and hexahistidine (HIS 6 ), which are particularly useful for isolation of the fusion polypeptide by affinity chromatography.
• relevant matrices for affinity chromatography are glutathione-, amylose-, and nickel- or cobalt-conjugated resins respectively.
• Many such matrices are available in "kit” form, such as the QIAexpressTM system (Qiagen) useful with (HIS 6 ) fusion partners and the Pharmacia GST purification system.
• the fusion partners also have protease cleavage sites, such as for Factor X a or Thrombin, which allow the relevant protease to partially digest the fusion polypeptide of the invention and thereby liberate the recombinant polypeptide of the invention therefrom.
• Fusion partners also include within their scope "epitope tags", which are usually short peptide sequences for which a specific antibody is available.
• epitope tags for which specific monoclonal antibodies are readily available include c-Myc, influenza virus haemagglutinin and FLAG tags.
• the step of introducing the expression vector into the host cell may be achieved by any suitable method including transfection, transduction of viral vectors, including adenoviral, modified lentiviral and other retroviral vectors, and transformation, the choice of which will be dependent on the host cell employed. Such methods are well known to those skilled in the art.
• Recombinant polypeptides may be produced by culturing a host cell transformed with the expression vector under conditions appropriate for protein expression, which will vary with the choice of expression vector and the host cell. This is easily ascertained by one skilled in the art through routine experimentation.
• Suitable host cells for expression may be prokaryotic or eukaryotic.
• One preferred host cell for expression of a polypeptide according to the invention is a bacterium.
• the bacterium used may be Escherichia coli.
• the host cell may be an insect cell such as, for example, SF9 cells that may be utilised with a baculovirus expression system.
• the antigen which may be administered with the LSD1 inhibitor, is in the form of a construct or vector from which it is expressible.
• the antigen can be synthesised using solution synthesis or solid phase synthesis as described, for example, by Atherton and Sheppard (1989, Solid Phase Peptide Synthesis: A Practical Approach, IRL Press at Oxford University Press, Oxford, England) or by Roberge et a/. (1995, Science, 269 : 202).
• the amino acids of the synthesised antigens can be non- naturally occurring or naturally occurring amino acids.
• unnatural amino acids and derivatives during peptide synthesis include but are not limited to, use of 4-amino butyric acid, 6- aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6- methylheptanoic acid, t-butylglycine, norleucine, norvaline, phenylglycine, ornithine, sarcosine, 2- thienyl alanine and/or D-isomers of amino acids.
• a list of unnatural amino acids contemplated by the present invention is shown in Table 3 ⁇ supra).
• the invention also contemplates modifying peptide antigens using ordinary molecular biological techniques so as to alter their resistance to proteolytic degradation or to optimise solubility properties or to render them more suitable as an immunogenic agent.
• Peptide antigens may be of any suitable size that can be utilized to stimulate or inhibit an immune response to a target antigen of interest. A number of factors can influence the choice of peptide size. For example, the size of a peptide can be chosen such that it includes, or corresponds to the size of, T cell epitopes and/or B cell epitopes, and their processing requirements.
• class I-restricted T cell epitopes are typically between 8 and 10 amino acid residues in length and if placed next to unnatural flanking residues, such epitopes can generally require 2 to 3 natural flanking amino acid residues to ensure that they are efficiently processed and presented.
• Class II-restricted T cell epitopes usually range between 12 and 25 amino acid residues in length and may not require natural flanking residues for efficient proteolytic processing although it is believed that natural flanking residues may play a role.
• class II-restricted epitopes generally contain a core of 9- 10 amino acid residues in the middle which bind specifically to class II M HC molecules with flanking sequences either side of this core stabilising binding by associating with conserved structures on either side of class II M HC antigens in a sequence independent manner.
• the functional region of class II-restricted epitopes is typically less than about 15 amino acid residues long.
• the size of linear B cell epitopes and the factors effecting their processing, like class II-restricted epitopes, are quite variable although such epitopes are frequently smaller in size than 15 amino acid residues.
• the size of the peptide is at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 amino acid residues.
• the size of the peptide is no more than about 500, 200, 100, 80, 60, 50, 40 amino acid residues.
• the size of the peptide is sufficient for presentation by an antigen-presenting cell of a T cell and/or a B cell epitope contained within the peptide.
• Criteria for identifying and selecting effective antigenic peptides can be found in the art. For example,
• the immune-modulating cell is an antigen-presenting cell, which presents an antigen corresponding to at least a portion of the target antigen.
• antigen-presenting cells include professional or facultative antigen-presenting cells.
• Professional antigen-presenting cells function physiologically to present antigen in a form that is recognised by specific T cell receptors so as to stimulate or anergise a T lymphocyte or B lymphocyte mediated immune response.
• Professional antigen-presenting cells not only process and present antigens in the context of the major histocompatability complex (M HC), but also possess the additional immunoregulatory molecules required to complete T cell activation or induce a tolerogenic response.
• M HC major histocompatability complex
• Professional antigen-presenting cells include, but are not limited to, macrophages, monocytes, B lymphocytes, cells of myeloid lineage, including monocytic-granulocytic-DC precursors, marginal zone Kupffer cells, microglia, T cells, Langerhans cells and dendritic cells including interdigitating dendritic cells and follicular dendritic cells.
• Non-professional or facultative antigen-presenting cells typically lack one or more of the immunoregulatory molecules required to complete T lymphocyte activation or anergy.
• non-professional or facultative antigen-presenting cells include, but are not limited to, activated T lymphocytes, eosinophils, keratinocytes, astrocytes, follicular cells, microglial cells, thymic cortical cells, endothelial cells, Schwann cells, retinal pigment epithelial cells, myoblasts, vascular smooth muscle cells, chondrocytes, enterocytes, thymocytes, kidney tubule cells and fibroblasts.
• the antigen- presenting cell is selected from monocytes, macrophages, B lymphocytes, cells of myeloid lineage, dendritic cells or Langerhans cells.
• the antigen-presenting cell expresses CDl lc and includes a dendritic cell.
• the antigen-presenting cell stimulates an immune response, including a pro-inflammatory immune response.
• Antigen-presenting cells for stimulating an immune response to an antigen or group of antigens to an antigen or group of antigens may be prepared according to any suitable method known to the skilled practitioner. Illustrative methods for preparing antigen-presenting cells for stimulating antigen-specific immune responses are described by Albert ef al. (International
• the antigen-presenting cells are isolated from a host, treated and then re-introduced or reinfused into the host.
• antigen-presenting cells can be obtained from the host to be treated either by surgical resection, biopsy, blood sampling, or other suitable technique. Such cells are referred to herein as "autologous" cells.
• the antigen-presenting cells or cell lines are prepared and/or cultured from a different source than the host. Such cells are referred to herein as "allogeneic" cells.
• allogeneic antigen-presenting cells or cell lines will share major and/or minor histocompatibility antigens to potential recipients (also referred to herein as "generic" antigen-presenting cells or cell lines).
• the generic antigen-presenting cells or cell lines comprise major histocompatibility (M HC) class I antigens compatible with a high percentage of the population (i.e. at least 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 92, 94 or 98%) that is susceptible or predisposed to a particular condition.
• M HC major histocompatibility
• the generic antigen-presenting cells or cell lines naturally express an immunostimulatory molecule as described herein, especially an immunostimulatory membrane molecule, at levels sufficient to trigger an immune response, desirably a T lymphocyte immune response (e.g. a cytotoxic T lymphocyte immune response), in the intended host.
• a T lymphocyte immune response e.g. a cytotoxic T lymphocyte immune response
• the antigen-presenting cells or cell lines are highly susceptible to treatment with at least one IFN as described herein and in International Pu blication No. WO 01/88097 (i.e. implied high level expression of class I HLA).
• antigen-presenting cells are made antigen-specific by a process including contacting or 'pulsing' the antigen-presenting cells with an antigen that corresponds to at least a portion of the target antigen for a time and under conditions sufficient to permit the antigen to be internalised by the antigen-presenting cells; and culturing the antigen-presenting cells so contacted for a time and under conditions sufficient for the antigen to be processed for
• the pulsed cells can then be used to stimulate autologous or allogeneic T cells in vitro or in vivo.
• the amount of antigen to be placed in contact with antigen-presenting cells can be determined empirically by a person of skill in the art.
• antigen-presenting cells are incubated with antigen for about 1 to 6 hr at 37° C.
• antigen for purified antigens and peptides, 0.1-10 ⁇ g/mL is suitable for producing antigen-specific antigen- presenting cells.
• the antigen should be exposed to the antigen-presenting cells for a period of time sufficient for those cells to internalise the antigen.
• the time and dose of antigen necessary for the cells to internalise and present the processed antigen may be determined using pulse-chase protocols in which exposure to antigen is followed by a washout period and exposure to a read-out system e.g. antigen reactive T cells.
• a protocol may be used to prepare cells and antigen for inducing tolerogenic responses.
• the length of time necessary for an antigen-presenting cell to present an antigen may vary depending on the antigen or form of antigen employed, its dose, and the antigen-presenting cell employed, as well as the conditions under which antigen loading is undertaken. These parameters can be determined by the skilled artisan using routine procedures.
• exogenous antigen to an antigen-presenting cell can be enhanced by methods known to practitioners in the art. For example, several different strategies have been developed for delivery of exogenous antigen to the endogenous processing pathway of antigen- presenting cells, especially dendritic cells. These methods include insertion of antigen into pH- sensitive liposomes (Zhou and Huang (1994), Immunomethods, 4: 229-235), osmotic lysis of pinosomes after pinocytic uptake of soluble antigen (Moore et al. (1988), Cell, 54: 777-785), coupling of antigens to potent adjuvants (Aichele et a/. (1990), J. Exp. Med.
• Recombinant bacteria e.g. E. coli
• transfected host mammalian cells may be pulsed onto dendritic cells (as particulate antigen, or apoptotic bodies respectively) for antigen delivery.
• VLPs Recombinant chimeric virus-like particles
• an antigen may be linked to, or otherwise associated with, a cytolysin to enhance the transfer of the antigen into the cytosol of an antigen-presenting cell of the invention for delivery to the M HC class I pathway.
• Exemplary cytolysins include saponin compounds such as saponin-containing Immune Stimulating Complexes (ISCOMs) (see, e.g., Cox and Coulter ( 1997), Vaccine 15(3) : 248-256 and U.S. Patent No. 6,352,697), phospholipases (see, e.g., Camilli et al. (1991), J. Exp. Med. , 173 : 751-754), pore-forming toxins (e.g. an a-toxin), natural cytolysins of gram-positive bacteria, such as listeriolysin 0 (LLO; e.g. Mengaud et al.
• ISCOMs saponin-containing Immune Stimulating Complexes
• LLO listeriolysin 0
• listeriolysin exhibits greater pore-forming ability at mildly acidic pH (the pH conditions within the phagosome), thereby facilitating delivery of vacuole (including phagosome and endosome) contents to the cytoplasm (see, e.g., Portnoy et al. (1992), Infect. Immun., 60 : 2710-2717).
• the cytolysin may be provided together with a pre-selected antigen in the form of a single composition or may be provided as a separate composition, for contacting the antigen- presenting cells.
• the cytolysin is fused or otherwise linked to the antigen, wherein the fusion or linkage permits the delivery of the antigen to the cytosol of the target cell.
• the cytolysin and antigen are provided in the form of a delivery vehicle such as, but not limited to, a liposome or a microbial delivery vehicle selected from virus, bacterium, or yeast.
• a delivery vehicle such as, but not limited to, a liposome or a microbial delivery vehicle selected from virus, bacterium, or yeast.
• the delivery vehicle is non-virulent.
• the delivery vehicle is a non-virulent bacterium, as for example described by Portnoy et al. in U.S. Patent No. 6,287,556, comprising a first polynucleotide encoding a non-secreted functional cytolysin operably linked to a regulatory polynucleotide which expresses the cytolysin in the bacterium, and a second polynucleotide encoding one or more pre-selected antigens.
• Non-secreted cytolysins may be provided by various mechanisms, e.g., absence of a functional signal sequence, a secretion incompetent microbe, such as microbes having genetic lesions (e.g.
• nonvirulent, non-pathogenic bacteria may be used; preferred microbes are relatively well characterised strains, particularly laboratory strains of E. coli, such as MC4100, MC1061, DH5a, etc.
• Other bacteria that can be engineered for the invention include well- characterized, nonvirulent, non-pathogenic strains of Listeria monocytogenes, Shigella flexneri, mycobacterium, Salmonella, Bacillus subtilis, etc.
• the bacteria are attenuated to be non-replicative, non-integrative into the host cell genome, and/or non-motile inter- or intra-cellularly.
• the delivery vehicles described above can be used to deliver one or more antigens to virtually any antigen-presenting cell capable of endocytosis of the subject vehicle, including phagocytic and non-phagocytic antigen-presenting cells.
• the subject methods generally require microbial uptake by the target cell and subsequent lysis within the antigen-presenting cell vacuole (including phagosomes and
• the antigen is produced inside the antigen-presenting cell by introduction of a suitable expression vector as, for example, described above.
• the antigen- encoding portion of the expression vector may comprise a naturally-occurring sequence or a variant thereof, which has been engineered using recombinant techniques.
• the codon composition of an antigen-encoding polynucleotide is modified to permit enhanced expression of the antigen in a target cell or tissue of choice using methods as set forth in detail in International Publications WO 99/02694 and WO 00/42215.
• codon-optimised polynucleotides at least one existing codon of a parent polynucleotide is replaced with a synonymous codon that has a higher translational efficiency in a target cell or tissue than the existing codon it replaces.
• the replacement step affects 5, 10, 15, 20, 25, 30%, more preferably 35, 40, 50, 60, 70% or more of the existing codons of a parent polynucleotide.
• the expression vector for introduction into the antigen-presenting cell will be compatible therewith such that the antigen-encoding polynucleotide is expressible by the cell.
• expression vectors of this type can be derived from viral DNA sequences including, but not limited to, adenovirus, adeno-associated viruses, herpes-simplex viruses and retroviruses such as B, C, and D retroviruses as well as spumaviruses and modified lentiviruses.
• Suitable expression vectors for transfection of animal cells are described, for example, by Wu and Ataai (2000, Curr. Opin. Biotechnol. 11(2) : 205-208), Vigna and Naldini (2000, J. Gene Med.
• the expression vector is introduced into the antigen-presenting cell by any suitable means which will be dependent on the particular choice of expression vector and antigen-presenting cell employed.
• suitable means of introduction are well- known to those skilled in the art. For example, introduction can be effected by use of contacting (e.g.
• the vectors are introduced by means of cationic lipids, e.g. liposomes.
• liposomes are commercially available (e.g. Lipofectin®, LipofectamineTM, and the like, supplied by Invitrogen, Waltham MA, USA).
• the antigen-specific antigen-presenting cells are obtained by isolating antigen-presenting cells or their precursors from a cell population or tissue to which modification of an immune response is desired.
• some of the isolated antigen-presenting cells or precursors will constitutively present antigens or have taken up such antigen in vivo that are targets or potential targets of an immune response for which stimulation or inhibition of an immune response is desired.
• the delivery of exogenous antigen is not essential.
• cells may be derived from biopsies of healthy or diseased tissues, lysed or rendered apoptotic and then pulsed onto antigen-presenting cells (e.g. dendritic cells).
• the antigen-presenting cells are cancer or tumor cells to which an antigen-specific immune response is required.
• cancers or tumor cells include cells of sarcomas and carcinomas e.g. fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
• lymphangiosarcoma lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymom
• the cancer or tumor cells will constitute facultative or non-professional antigen-presenting cells, and may in some instances require further modification to enhance their antigen-presenting functions.
• the antigen- presenting cells are further modified to express one or more immunoregulatory molecules, which include any molecules occurring naturally in animals that may regulate or directly influence immune responses including : proteins involved in antigen processing and presentation such as TAP1/TAP2 transporter proteins, proteosome molecules such as LM P2 and LMP7, heat shock proteins such as gp96, HSP70 and HSP90, and major histocompatibility complex (M HC) or human leucocyte antigen (HLA) molecules; factors that provide co-stimulation signals for T cell activation such as B7 and CD40; factors that provide co-inhibitory signals for direct killing of T cells or induction of T lymphocyte or B lymphocyte anergy or stimulation of T regulatory cell (Treg) generation such as OX-2, PD-L1 or PD-L2; accessory
• TAP1/TAP2 transporter proteins proteins involved
• lymphokines and cytokines such as interferons ⁇ , ⁇ and ⁇ , interleukins (e.g., IL-2, IL-7, IL-12, IL- 15, IL-22, etc.), factors stimulating cell growth (e.g. GM-SCF) and other factors (e.g. tumor necrosis factors (TNFs), DC-SIGN, ⁇ , ⁇ and transforming growth factor- ⁇ (TGF- ⁇ ).
• the immunoregulatory molecules are selected from a B7 molecule (e.g. B7-1, B7-2 or B7-3) and an ICAM molecule (e.g. ICAM-1 and ICAM-2).
• antigen-presenting cells expressing the desired immunostimulatory molecule(s) may be isolated or selected from a heterogeneous population of cells. Any method of isolation/selection is contemplated by the present invention, examples of which are known to those of skill in the art. For instance, one can take advantage of one or more particular characteristics of a cell to specifically isolate that cell from a heterogeneous population.
• Such characteristics include, but are not limited to, anatomical location of a cell, cell density, cell size, cell morphology, cellular metabolic activity, cell uptake of ions such as Ca 2+ , K + , and H + ions, cell uptake of compounds such as stains, markers expressed on the cell surface, protein fluorescence and membrane potential.
• Suitable methods that can be used in this regard include surgical removal of tissue, flow cytometry techniques such as fluorescence- activated cell sorting (FACS), immunoaffinity separation (e.g. magnetic bead separation such as DynabeadTM separation), density separation (e.g. metrizamide, PercollTM, or FicollTM gradient centrifugation), and cell-type specific density separation.
• the cells are isolated by flow cytometry or by immunoaffinity separation using an antigen-binding molecule that is immuno- interactive with the immunoregulatory molecule.
• the immunoregulatory molecule can be provided to the antigen-presenting cells in soluble form.
• the immunoregulatory molecule is a B7 molecule that lacks a functional transmembrane domain (e.g. that comprises a B7 extracellular domain), non-limiting examples of which are described by McHugh et al. (1998, Clin. Immunol. Immunopathol., 87(1) : 50-59), Faas et al. (2000, J. Immunol. , 164(12) : 6340-6348) and Jeannin et a/. (2000, Immunity, 13(3) : 303-312).
• the immunoregulatory molecule is a B7 molecule that lacks a functional transmembrane domain (e.g. that comprises a B7 extracellular domain), non-limiting examples of which are described by McHugh et al. (1998, Clin. Immunol. Immunopathol., 87(1) : 50-59), Faas et
• immunostimulatory protein is a B7 derivative including, but not limited to, a chimeric or fusion protein comprising a B7 molecule, or biologically active fragment thereof, or variant or derivative of these, linked together with an antigen binding molecule such as an immunoglobulin molecule or biologically active fragment thereof.
• a polynucleotide encoding the amino acid sequence corresponding to the extracellular domain of the B7-1 molecule, containing amino acids from about position 1 to about position 215, is joined to a polynucleotide encoding the amino acid sequences corresponding to the hinge, CH2 and CH3 regions of human Ig Cyl, using PCR, to form a construct that is expressed as a B7Ig fusion protein.
• DNA encoding the amino acid sequence corresponding to a B7Ig fusion protein has been deposited with the American Type Culture
• a soluble immunoregulatory molecule may be prolonged by any suitable procedure if desired.
• such molecules are chemically modified with polyethylene glycol (PEG), including monomethoxy-polyethylene glycol, as for example disclosed by Chapman et al (1999, Nature Biotechnology, 17 : 780-783).
• PEG polyethylene glycol
• the antigen-presenting cells are cultured in the presence of at least one interferon for a time and under conditions sufficient to enhance the antigen presenting function of the cells, and washing the cells to remove the interferon(s).
• the step of culturing may comprise contacting the cells with at least one type I interferon and/or a type II interferon.
• the at least one type I interferon is suitably selected from the group consisting of an IFN-a, an IFN- ⁇ , a biologically active fragment of an IFN-a, a biologically active fragment of an IFN- ⁇ , a variant of an IFN-a, a variant of an IFN- ⁇ , a variant of a said biologically active fragment, a derivative of an IFN-a, a derivative of an IFN- ⁇ , a derivative of a said biologically active fragment, a derivative of a said variant, an analogue of IFN-a and an analogue of IFN- ⁇ .
• the type II interferon is selected from the group consisting of an IFN- Y, a biologically active fragment of an IFN- ⁇ , a variant of an IFN- ⁇ , a variant of said biologically active fragment, a derivative of an IFN- ⁇ , a derivative of said biologically active fragment, a derivative of said variant and an analogue of an IFN- ⁇ .
• exemplary methods and conditions for enhancing the antigen-presenting functions of antigen-presenting cells using interferon treatment are described in International Publication No. WO 01/88097.
• the antigen-presenting cells e.g. cancer cells
• cell lines are suitably rendered inactive to prevent further proliferation once administered to the subject.
• Any physical, chemical, or biological means of inactivation may be used, including but not limited to irradiation (generally with at least about 5,000 cGy, usually at least about 10,000 cGy, typically at least about 20,000 cGy) ; or treatment with mitomycin-C (usually at least 10 pg/mL; more usually at least about 50 g/mL).
• the antigen-presenting cells may be obtained or prepared to contain and/or express one or more antigens by any number of means, such that the antigen(s) or processed form(s) thereof, is (are) presented by those cells for potential modulation of other immune cells, including T lymphocytes and B lymphocytes, and particularly for producing T lymphocytes and B lymphocytes that are primed to respond to a specified antigen or group of antigens.
• the antigen-presenting cell is an immune-effector cell.
• the antigen-presenting cells described above are useful for producing primed T lymphocytes to an antigen or group of antigens.
• the antigen-specific antigen-presenting cells are useful for producing T lymphocytes that exhibit tolerance/anergy to an antigen or group of antigens.
• the efficiency of inducing lymphocytes, especially T lymphocytes, to exhibit an immune response or tolerance/anergy to a specified antigen can be determined by any suitable method including, but not limited to, assaying T lymphocyte cytolytic activity in vitro using for example antigen-specific antigen-presenting cells as targets of antigen-specific cytolytic T lymphocytes (CTL) ; assaying antigen-specific T lymphocyte proliferation (see, e.g., Vollenweider and Groseurth ( 1992), J. Immunol. Meth.
• CTL antigen-specific cytolytic T lymphocytes
• the present invention also provides antigen-specific B or T lymphocytes, especially T lymphocytes, which respond in an antigen-specific fashion to representation of the antigen.
• antigen-specific T lymphocytes are produced by contacting an antigen-presenting cell as defined above with a population of T lymphocytes, which may be obtained from any suitable source such as spleen or tonsil/lymph nodes but is preferably obtained from peripheral blood.
• the T lymphocytes can be used as crude preparations or as partially purified or substantially purified preparations, which are suitably obtained using standard techniques as, for example, described in "Immunochemical Techniques, Part G: Separation and Characterization of Lymphoid Cells" (1984, Meth. in Enzymol.
• T lymphocytes are contacted with antigen-specific antigen-presenting cells as described herein for an adequate period of time for priming or anergising the T
• lymphocytes to the antigen or antigens presented by those antigen-presenting cells. This period will usually be at least about 1 day, and up to about 5 days.
• the antigen-specific anergy induced by such cells desirably involves the induction of one or more types of antigen-specific regulatory lymphocytes, especially regulatory T lymphocytes.
• regulatory T lymphocytes especially regulatory T lymphocytes.
• Several populations of regulatory T lymphocytes are known to inhibit the response of other (effector) lymphocytes in an antigen-specific manner including, for example, Trl
• Th3 lymphocytes Th3 lymphocytes
• Th2 lymphocytes Th2 lymphocytes
• Trl lymphocytes can emerge after several rounds of stimulation of human blood T cells by allogeneic monocytes in the presence of IL- 10. This subpopulation secretes high levels of IL-10 and moderate levels of TGFP but little IL-4 or IFNy (Groux et a/. (1997), Nature, 389 : 737-742).
• the Th3 regulatory subpopulation refers to a specific subset induced following antigen delivery via the oral (or other mucosal) route. They produce predominantly TGF0, and only low levels of IL-10, IL-4 or IFNy, and provide specific help for IgA production (Weiner ef al. (2001), Microbes Infect, 3 : 947-954). They are able to suppress both Th l and Th2-type effector T cells.
• Th2 lymphocytes produce high levels of IL-4, IL-5 and IL-10 but low IFNy and TGF0. Th2 lymphocytes are generated in response to a relative abundance of IL-4 and lack of IL-12 in the environment at the time of presentation of their cognate peptide ligands (O'Garra and Arai (2000), Trends Cell Biol, 10 : 542-550). T lymphocyte signalling by CD86 may also be important for generation of Th2 cells (Lenschow et al. (1996), Immunity, 5 : 285-293; Xu et al. (1997), J
• CD8 + CD28 " regulatory or "suppressor" subset of T lymphocytes can be induced by repetitive antigenic stimulation in vitro. They are M HC class I-restricted, and suppress CD4 + T cell responses.
• CD4 + CD25 + regulatory or "suppressor" subset of T lymphocytes inhibit a variety of autoimmune and inflammatory diseases and they are also efficient in the suppression of alloantigen responses.
• these lymphocytes can down-regulate the immune response by affecting T cell responses, antibody production, cytokine secretion and antigen-presenting cells (see, e.g., Suvas et a/. (2003), J Exp Med. , 198(6) : 889-901 ; Taams et al. (2003), Transpl Immunol. , 11(3- 4) : 277-85; Jonuleit et al. (2003), Transpl Immunol.
• CD4+CD25+ regulatory T cells are generated by repetitive antigenic stimulation in vitro (Feunou et al. (2003), J Immunol., 171(7) : 3475-84).
• NK T lymphocytes which express the NK cell marker, CD161, and whose TCR are Va24JaQ in human and Va l4Ja281 in mouse, are activated specifically by the non-polymorphic CDld molecule through presentation of a glycolipid antigen (Kawano et a/. (1997), Science, 278:
• ⁇ T lymphocytes have been implicated in the downregulation of immune responses in various inflammatory diseases and in the suppression of inflammation associated with induction of mucosal tolerance.
• the tolerance induced by mucosal antigen was transferable to untreated recipient mice by small numbers of ⁇ T cells (McMenamin ef al. (1995), J Immunol, 154: 4390- 4394; McMenamin et al. (1994), Science, 265 : 1869-1871).
• mucosal tolerance induction was blocked by the administration of the GL3 antibody that blocks ⁇ T cell function (Ke et al. (1997), J Immunol, 158 : 3610-3618).
• the antigen-specific T lymphocytes are produced in contact with antigen- presenting cells in vitro or in vivo, the antigen-specific anergy induced by the antigen-presenting cells reflects the inability of the antigen-specific lymphocytes to respond to subsequent
• antigen-specific lymphocytes are suitably provided.
• IL-10 is a cytokine with potent immunosuppressive properties. IL-10 inhibits antigen-specific T lymphocyte proliferation at different levels. IL-10 inhibits the antigen-presenting and accessory cell function of professional antigen-presenting cells such as monocytes, dendritic cells and Langerhans cells by downregulation of the expression of MHC class II molecules and of the adhesion and co-stimulatory molecules ICAM-1 and B7.1 and B7.2 (reviewed in Interleukin 10 (1995), de Vries and de Waal Malefyt, eds., Austin Tex.). IL-10 also inhibits IL-12 production by these cells.
• IL-12 promotes T lymphocyte activation and the differentiation of Th l lymphocytes (D'Andrea, et al. (1993), J. Exp. Med., 178 : 1041-1048; Hsieh et al. (1993), Science, 260 : 547-549).
• IL-10 directly inhibits T lymphocyte proliferation by inhibiting IL-2 gene transcription and IL-2 production by these cells (reviewed in Interleukin 10 (1995), de Vries and de Waal Malefyt, eds., Austin Tex.), and itself promotes antigen-presenting cells that induce regulatory T cells (U.S.
• the presence of anergic T lymphocytes may be determined by assaying IL-10 production, e.g. by ELISA in cell supernatants, or by flow cytometric analysis of intracellular staining.
• the immune-modulating agent is an antigen-binding molecule that is immuno-interactive with the target antigen.
• the target antigen is expressed in a disease or condition or by a specific pathogen for which an enhanced immune response is required.
• the target antigen is aberrantly expressed, typically at a higher level in the disease or condition as compared to the normal state or to a state in which the disease or condition is absent.
• the antigen-binding molecule is suitably interactive with a target antigen as previously described. Numerous antigen-binding molecules useful in the present invention are known in the art.
• the antigen-binding molecule is immuno-interactive with an antigen selected from the Cripto-lprotein, Pim-1 protein or an antigen present in a colon cancer cell lysate, as disclosed, for example, in United States Patent Application Publication No. 20040176576.
• the antigen-binding molecule is an antibody, especially a whole polyclonal antibody.
• Such antibodies may be prepared, for example, by injecting an antigen that corresponds to at least a portion of the target antigen into a production species, which may include mice or rabbits, to obtain polyclonal antisera. Methods of producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols which may be used are described for example in Coligan et al. (1991), Current Protocols in Immunology, (John Wiley & Sons, Inc), and Ausubel et al. (1998, supra), in particular Section III of Chapter 11.
• monoclonal antibodies may be produced using the standard method as described, for example, by Kohler and M ilstein (1975, Nature, 256: 495-497), or by more recent modifications thereof as described, for example, in Coligan et al. (1991, supra) by immortalising spleen or other antibody producing cells derived from a production species which has been inoculated with one or more antigens as described above.
• the invention also contemplates as antigen-binding molecules Fv, Fab, Fab' and F(ab') 2 immunoglobulin fragments.
• the antigen-binding molecule may comprise a synthetic stabilised Fv fragment.
• Exemplary fragments of this type include single chain Fv fragments (sFv, frequently termed scFv) in which a peptide linker is used to bridge the N-terminus or C-terminus of a V H domain with the C-terminus or N-terminus, respectively, of a ⁇ J L domain.
• sFv single chain Fv fragments
• scFv single chain Fv fragments
• ScFv lack all constant parts of whole antibodies and are not able to activate complement.
• ScFvs may be prepared, for example, in accordance with methods outlined in Kreber et a/ (1997, J. Immunol. Methods, 201(1) : 35-55). Alternatively, they may be prepared by methods described in U.S. Patent No 5,091,513, European Patent No 239,400 or the articles by Winter and M ilstein (1991, Nature, 349 : 293) and PlCickthun et al (1996, In Antibody engineering: A practical approach., 203-252).
• the synthetic stabilised Fv fragment comprises a disulfide stabilised Fv (dsFv) in which cysteine residues are introduced into the V H and ⁇ J L domains such that in the fully folded Fv molecule the two residues will form a disulfide bond between them.
• dsFv disulfide stabilised Fv
• Suitable methods of producing dsFv are described for example in Glockscuther et al. (1990), Biochem. , 29 : 1362-1367; Reiter et al. (1994), J. Biol. Chem., 269 : 18327-18331; Reiter et al. (1994), Biochem. 33 : 5451-5459; Reiter et a/. (1994), Cancer Res. , 54: 2714-2718; Webber et a/. (1995), Mol. Immunol. , 32 : 249- 258.
• dsFv disulfide stabilised Fv
• the proteinaceous molecules of the invention are useful in methods for enhancing an immune response in a subject to a target antigen by an immune- modulating agent and inhibiting PD-L1 and/or PD-L2 activity, including nuclear translocation of PD- LI and/or PD-L2. Furthermore, the proteinaceous molecules of the invention are useful in methods for the treatment or prevention of a condition involving LSDl and/or PKC overexpression, such as a cancer.
• the proteinaceous molecules of the invention are useful for the inhibition of LSDl nuclear translocation.
• the proteinaceous molecules of the invention are useful in methods for altering at least one of formation, proliferation,
• the proteinaceous molecules of the invention are useful for inhibiting the proliferation, survival or viability of an LSDl overexpressing cell.
• the proteinaceous molecules of the invention are useful for the treatment or prevention of a condition involving LSDl overexpression in a subject, such as a cancer, especially breast cancer.
• the proteinaceous molecule of the invention for therapy, or in the manufacture of a medicament for therapy.
• the present invention also encompasses a proteinaceous molecule of the invention for use in therapy, or for use as a medicament.
• the proteinaceous molecules of the invention are useful for the inhibition of LSDl, particularly the nuclear translocation of LSDl.
• the proteinaceous molecules of the invention are useful in methods of inhibiting an activity of LSDl, such as the nuclear translocation of LSDl or the phosphorylation of LSDl.
• the proteinaceous molecules of the invention will, as a result of their inhibitory action on LSDl, be useful in methods of altering at least one of (i) formation ; (ii) proliferation ; (iii) maintenance; (iv) EMT; or (v) M ET of an LSDl overexpressing cell.
• the proteinaceous molecule of the invention results in a reduction, impairment, abrogation or prevention of the (i) formation ; (ii) proliferation; (iii) maintenance; or (iv) EMT of an LSDl overexpressing cell; and/or in the enhancement of (v) M ET of an LSDl overexpressing cell.
• the proteinaceous molecules of the invention may be used for treating or preventing a cancer in a subject, wherein the cancer comprises at least one LSDl overexpressing cell.
• the cancer may comprise cancer stem cells and non-cancer stem cell tumor cells.
• the cancer is selected from breast, prostate, lung, bladder, pancreatic, colon, melanoma, retinoblastoma, liver or brain cancer; especially breast cancer.
• the proteinaceous molecules of the invention are used for treating, preventing and/or relieving the symptoms of a malignancy, particularly a metastatic cancer.
• the proteinaceous molecules of the invention are used for treating, preventing and/or relieving the symptoms of a metastatic cancer.
• metastatic cancer examples include, but are not limited to, metastatic breast, prostate, lung, bladder, pancreatic, colon, melanoma, retinoblastoma, liver or brain cancer.
• the brain cancer is a glioma.
• the metastatic cancer is metastatic breast cancer.
• the proteinaceous molecules are useful in methods involving LSDl overexpressing cells.
• the LSDl overexpressing cell is selected from a breast, prostate, testicular, lung, bladder, pancreatic, colon, melanoma, leukemia, retinoblastoma, liver or brain cell; especially a breast cell.
• the LSDl overexpressing cell is a breast epithelial cell, especially a breast ductal epithelial cell.
• the LSDl overexpressing cell is a cancer stem cell or a non- cancer stem cell tumor cell; especially a cancer stem cell tumor cell; most especially a breast cancer stem cell tumor cell.
• the cancer stem cell tumor cell expresses CD24 and CD44, particularly CD44 high , CD24 low .
• the methods further comprise detecting overexpression of an
• the LSDl gene in a tumor sample obtained from the subject, wherein the tumor sample comprises the cancer stem cell tumor cells and optionally the non-cancer stem cell tumor cells, prior to administering the proteinaceous molecule of the invention to the subject.
• the proteinaceous molecules of the invention are suitable for treating an individual who has been diagnosed with a cancer, who is suspected of having a cancer, who is known to be susceptible and who is considered likely to develop a cancer, or who is considered to develop a recurrence of a previously treated cancer.
• the cancer may be hormone receptor negative.
• the cancer is hormone receptor negative and is, thus, resistant to hormone or endocrine therapy.
• the breast cancer is hormone receptor negative.
• the breast cancer is estrogen receptor negative and/or progesterone receptor negative.
• the proteinaceous molecule of the invention may be useful. Accordingly, in another aspect of the invention, there is provided the use of a proteinaceous molecule of the invention for treating or preventing a condition in a subject in respect of which LSD1 inhibition is associated with effective treatment. The invention also contemplates a method of treating or preventing a condition in a subject in respect of which LSD1 inhibition is associated with effective treatment. In a further aspect of the invention, there is provided the use of a proteinaceous molecule of the invention in the manufacture of a medicament for treating or preventing a condition in a subject in respect of which LSD1 inhibition is associated with effective treatment. The invention also provides a proteinaceous molecule of the invention for use in treating or preventing a condition in respect of which LSD1 inhibition is associated with effective treatment.
• Non-limiting examples of conditions involving LSD1 overexpression or activity and, thus, conditions in which LSD1 inhibition is associated with effective treatment include cancer; sickle cell disease; viral infection such as HIV, herpes simplex virus (e.g. HSV-1 or HSV-2), adenovirus, human papillomavirus, parvovirus, smallpox virus, vaccinia virus, hepadnaviridae, polyoma virus, Epstein-Barr virus, hepatitis virus (e.g. hepatitis B virus), or varicella-zoster virus infection;
• herpes simplex virus e.g. HSV-1 or HSV-2
• adenovirus e.g. HSV-1 or HSV-2
• adenovirus e.g. HSV-1 or HSV-2
• human papillomavirus papillomavirus
• parvovirus smallpox virus
• smallpox virus vaccinia virus
• hepadnaviridae
• inflammatory conditions such as atherosclerosis, a respiratory inflammatory disorder (e.g.
• respiratory distress syndrome asthma, chronic obstructive pulmonary disease, bronchial hyperresponsiveness, bronchoconstriction, airway inflammation, airway remodelling or cystic fibrosis), chronic inflammatory bowel disease, ulcerative colitis, Crohn's disease, a chronic skin inflammatory disease (e.g.
• psoriasis or atopic dermatitis mesangial glomerulonephritis
• Kawasaki disease disseminated intravascular inflammation
• Caffey disease twin reversed arterial perfusion syndrome
• allergic vasculitis arthritis
• vasculitis coronary artery disease
• carotid artery disease transplant vasculopathy
• rheumatoid arthritis hepatic cirrhosis
• nephritis cardiovascular conditions such as thrombosis, Budd-Chiari syndrome, Paget-Schroetter disease, myocardial infarction, coronary heart disease, coronary artery disease, stroke, heart failure or hypertension
• neurological disorders such as schizophrenia, developmental disorders, depression, epilepsy, drug addiction or neurodegenerative diseases (e.g. Parkinson's disease, Huntington's disease,
• the condition is cancer.
• the methods involve the administration of a further active agent as described in Section 3 supra, such as an additional cancer therapy and/or an anti-infective agent. 6.
• a further active agent as described in Section 3 supra, such as an additional cancer therapy and/or an anti-infective agent. 6.
• the amino acid sequence corresponding to residues 108 to 118 of LSD1 comprises a potential phosphorylation site on serine 111.
• the proteinaceous molecules of the invention may bind to a PKC, especially PKC- ⁇ , thereby inhibiting the phosphorylating activity of said PKC and, consequently, inhibiting the phosphorylation of LSD1.
• the lack of LSD1 phosphorylation is proposed to inhibit the nuclear translocation of LSD1.
• the proteinaceous molecules of the invention are useful for inhibiting the phosphorylating activity of a PKC, such as inhibiting PKC phosphorylation of LSD1 and altering at least one of formation, proliferation, maintenance, EMT or MET of a PKC overexpressing cell.
• proteinaceous molecules of the invention are useful for the treatment or prevention of a condition involving PKC overexpression in a subject, such as a cancer.
• the PKC is PKC- ⁇ .
• the proteinaceous molecules of the invention selectively inhibit the phosphorylating activity of PKC- ⁇ over at least one other PKC enzyme or isoform, such as PKC- a, PKC- ⁇ , PKC-Y, PKC- ⁇ , PKC- ⁇ , PKC- ⁇ , PKC- ⁇ , PKC- ⁇ or PKC-v.
• the proteinaceous molecules of the invention selectively inhibit the phosphorylating activity of PKC- ⁇ over the other 10 PKC enzymes.
• the proteinaceous molecules of the invention exhibit PKC- ⁇ selectivity of greater than about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or greater than about 100-fold with respect to inhibition of the phosphorylating activity of another PKC (i.e. a PKC other than PKC- ⁇ , such as PKC-a, PKC- ⁇ , PKC- ⁇ , PKC- ⁇ , PKC- ⁇ , PKC- ⁇ , PKC- ⁇ , PKC- ⁇ , PKC- ⁇ or PKC-v).
• PKC- ⁇ selectivity of greater than about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or greater than about 100-fold with respect to inhibition of the phosphorylating activity of another PKC (i.e. a PKC other than PKC- ⁇ , such as PKC-a, PKC- ⁇ , PKC- ⁇ , PKC- ⁇ , PKC- ⁇ , PKC- ⁇ , PKC- ⁇ , PKC- ⁇ or PKC-v).
• selective molecules display at least 50-fold greater inhibition towards PKC-
• selective molecules display at least 500-fold greater inhibition towards PKC- ⁇ than towards another PKC. In yet further embodiments, selective molecules display at least 100-fold greater inhibition towards PKC- ⁇ than towards another PKC.
• the proteinaceous molecules of the invention are non-selective inhibitors of the phosphorylating activity of PKC- ⁇ .
• the proteinaceous molecules of the invention are useful in methods of altering at least one of (i) formation; (ii) proliferation ; (iii) maintenance; (iv) EMT; or (v) M ET of a PKC
• the cell is contacted with a formation-, proliferation-, maintenance- , EMT- or MET-modulating amount of a proteinaceous molecule of the invention.
• the proteinaceous molecule of the invention results in a reduction, impairment, abrogation or prevention of the (i) formation ; (ii) proliferation ; (iii) maintenance; or (iv) EMT of a PKC overexpressing cell; and/or in the enhancement of (v) M ET of a PKC overexpressing cell.
• the proteinaceous molecules of the invention may be used for treating or preventing a cancer in a subject, wherein the cancer comprises at least one PKC overexpressing cell.
• the PKC is PKC- ⁇ .
• the cancer may be selected from, but is not limited to, breast, prostate, lung, bladder, pancreatic, colon, melanoma, retinoblastoma, liver or brain cancer; especially breast cancer.
• the cancer is a metastatic cancer, especially a metastatic breast cancer.
• the proteinaceous molecules of the invention are used for treating, preventing and/or relieving the symptoms of a malignancy, particularly a metastatic cancer. In preferred embodiments, the proteinaceous molecules of the invention are used for treating, preventing and/or relieving the symptoms of a metastatic cancer.
• the PKC is PKC- ⁇ .
• Suitable PKC- ⁇ overexpressing cells may include, but are not limited to, breast, prostate, lung, bladder, pancreatic, colon, melanoma, liver, retina or glioma cells; especially breast cells.
• overexpressing cell is a breast epithelial cell, especially a breast ductal epithelial cell.
• the PKC- ⁇ overexpressing cell is a cancer stem cell or a non- cancer stem cell tumor cell ; preferably a cancer stem cell tumor cell.
• the CSC tumor cell expresses CD24 and CD44, particularly CD44 high , CD24 low .
• the proteinaceous molecules of the invention are suitable for treating an individual who has been diagnosed with a cancer, who is suspected of having a cancer, who is known to be susceptible and who is considered likely to develop a cancer, or who is considered to develop a recurrence of a previously treated cancer.
• the cancer may be hormone receptor negative.
• the cancer is hormone receptor negative and is, thus, resistant to hormone or endocrine therapy.
• the breast cancer is hormone receptor negative.
• the breast cancer is estrogen receptor negative and/or progesterone receptor negative.
• the proteinaceous molecule of the invention may be useful. Accordingly, in another aspect of the invention, there is provided the use of a proteinaceous molecule of the invention for treating or preventing a condition in a subject in respect of which PKC inhibition is associated with effective treatment. The invention also contemplates a method of treating or preventing a condition in a subject in respect of which PKC inhibition, particularly PKC- ⁇ inhibition, is associated with effective treatment. In a further aspect of the invention, there is provided the use of a proteinaceous molecule of the invention in the manufacture of a medicament for treating or preventing a condition in a subject in respect of which PKC inhibition is associated with effective treatment. The invention also provides a proteinaceous molecule of the invention for use in treating or preventing a condition in respect of which PKC inhibition is associated with effective treatment.
• Conditions involving PKC overexpression or activity, particularly PKC- ⁇ overexpression or activity, and, thus, conditions in which PKC inhibition is associated with effective treatment include, but are not limited to, cancer; neurological and vascular disorders such as Down's syndrome, memory and cognitive impairment, dementia, amyloid neuropathies, brain
• bronchitis obstructive bronchitis
• spastic bronchitis allergic bronchitis
• allergic bronchitis allergic asthma
• bronchial asthma emphysema or chronic obstructive pulmonary disease (COPD)
• cardiac disorders such as heart failure, atherosclerosis, cardiac fibrosis, hypertrophy or ischemic heart disease
• dermatoses such as psoriasis, toxic and allergic contact eczema, atopic eczema, seborrheic eczema, lichen simplex, sunburn, pruritis in the anogenital area, alopecia areata, hypertrophic scars, discoid lupus erythematosus, follicular and wide-area pyodermias, end
• the methods further comprise detecting overexpression of a PKC gene, especially a PKC- ⁇ gene, in a tumor sample obtained from the subject, wherein the tumor sample comprises the cancer stem cell tumor cells and optionally the non-cancer stem cell tumor cells, prior to administering the proteinaceous molecule of the invention to the subject.
• a PKC gene especially a PKC- ⁇ gene
• the methods involve the administration of a further active agent as described in Section 3 supra, such as an additional cancer therapy and/or an anti-infective agent.
• a skilled person would be well aware of suitable assays used to evaluate LSD1, PDL-1, PDL-2 and/or PKC inhibition, such as inhibition of nuclear translocation and phosphorylating activity, and to identify proteinaceous molecules that are LSD1 or PKC inhibitors. Screening for active agents according to the invention can be achieved by any suitable method.
• the method may include contacting a cell expressing a polynucleotide corresponding to a gene that encodes LSD1, PKC, PD-L1 and/or PD-L2 with an agent suspected of having the inhibitory activity and screening for the inhibition of the level or functional activity of LSD1, PKC, PD-L1 and/or PD- L2, or the lowering of the level of a transcript encoded by the polynucleotide, or the inhibition of the activity or expression of a downstream cellular target of the polypeptide or of the transcript (hereafter referred to as target molecules).
• target molecules a downstream cellular target of the polypeptide or of the transcript
• Detecting such inhibition can be achieved utilizing techniques including, but not restricted to, ELISA, cell-based ELISA, inhibition ELISA, Western blots, immunoprecipitation, immunofluorescence, slot or dot blot assays, immunostaining, RIA, scintillation proximity assays, fluorescent immunoassays using antigen-binding molecule conjugates or antigen conjugates of fluorescent substances such as fluorescein or rhodamine, Ouchterlony double diffusion analysis, immunoassays employing an avidin-biotin or a streptavidin- biotin detection system, and nucleic acid detection assays including reverse transcriptase polymerase chain reaction (RT-PCR).
• ELISA enzyme-binding
• cell-based ELISA Western blots
• immunoprecipitation immunofluorescence
• slot or dot blot assays immunostaining
• RIA scintillation proximity assays
• a polynucleotide from which a LSD1, PKC, PD-L1 and/or PD-L2 is regulated or expressed may be naturally occurring in the cell which is the subject of testing or it may have been introduced into the host cell for the purpose of testing.
• the naturally- occurring or introduced polynucleotide may be constitutively expressed, thereby providing a model useful in screening for agents which down-regulate expression of an encoded product of the sequence wherein the down regulation can be at the nucleic acid or expression product level.
• a polynucleotide may comprise the entire coding sequence that codes for LSD1, PKC, PD-L1 and/or PD-L2 or it may comprise a portion of that coding sequence (e.g. the active site of LSDl, PKC, PD-Ll and/or PD-L2) or a portion that regulates expression of the corresponding gene that encodes LSDl, PKC, PD-Ll and/or PD-L2 (e.g. a promoter).
• the promoter that is naturally associated with the polynucleotide may be introduced into the cell that is the subject of testing.
• detecting modulation of the promoter activity can be achieved, for example, by operably linking the promoter to a suitable reporter polynucleotide including, but not restricted to, green fluorescent protein (GFP), luciferase, ⁇ -galactosidase and catecholamine acetyl transferase (CAT). Modulation of expression may be determined by measuring the activity associated with the reporter polynucleotide.
• GFP green fluorescent protein
• CAT catecholamine acetyl transferase
• These methods provide a mechanism for performing high throughput screening of putative modulatory agents such as proteinaceous or non-proteinaceous agents comprising synthetic, combinatorial, chemical and natural libraries. These methods will also facilitate the detection of agents which bind either the polynucleotide encoding the target molecule or which inhibit the expression of an upstream molecule, which subsequently inhibits the expression of the polynucleotide encoding the target molecule. Accordingly, these methods provide a mechanism of detecting agents that either directly or indirectly inhibit the expression or activity of a target molecule according to the invention.
• test agents are screened using commercially available assays, illustrative examples of which include EpiQuik Histone Demethylase LSDl Inhibitor Screening Assay Kit (Epigentek Group, Brooklyn, USA), the LSDl Inhibitor Screening Assay Kit (Cayman Chemical Company, Ann Arbor, USA), the PKC Kinase Activity Assay Kit (Abeam, Cambridge, United Kingdom), Protein Kinase C Assay Kit (Panvera Corporation, Madison, USA), and Cell-based Immune-checkpoint Assays (Genscript, Piscataway, USA).
• Compounds may be further tested in the animal models to identify those compounds having the most potent in vivo effects. These molecules may serve as "lead compounds" for the further development of pharmaceuticals by, for example, subjecting the compounds to sequential modifications, molecular modeling, and other routine procedures employed in rational drug design.
• LSD1 peptide inhibitors LI, L2 and L3 (refer to Table 4) were synthesized using automated modern solid phase peptide synthesis and purification technology using the mild Fmoc chemistry method, for example, as described in Ensenat-Waser, et a/. (2002) IUBMB Life, 54: 33- 36 and WO 2002/010193. Couplings were performed using standard ⁇ -diisopropylcarbodiimide (DIC)/hydroxybenzotriazole (HOBt) coupling. Following deprotection, peptides were purified using automated preparative reversed phase-high performance liquid chromatography (RP-HPLC).
• RP-HPLC reversed phase-high performance liquid chromatography
• All peptides tested were myristoylated through the N-terminal amino group of the N- terminal amino acid. Myristoylation was carried out by covalently coupling myristic acid to the N - terminal residue using standard DIC/ HOBt coupling as described above, prior to deprotection and purification of the peptides.
• PD-Ll traditionally being described as a cell-surface signaling protein
• microscopic analysis demonstrated that PD-Ll has a clear nuclear signal in MCF7 and MDA-MB-231 cells ( Figure 1).
• PD-Ll has a significantly more nuclear distribution in the mesenchymal MCF7 and the more aggressive triple negative MDA-MB-231 breast cancer cell lines.
• Fn/c the ratio of nuclear to cytoplasmic fluorescence
• TNFI total nuclear fluorescence
• LSDls111p LSD1 phosphorylated at serine 111
• MCF7ST mesenchymal stimulated MCF7 cells
• MDA-MB-231 MDA-MB-231
• the PCC co- localization coefficient which measures the degree of co-localization of two proteins within the nucleus of a cell
• MCF7NS epithelial non-stimulated MCF7 cells
• the highest PCC was in the MDA-MB-231 cells. This data indicates a direct relationship between LSDls111p and PD-Ll in the nucleus of a cancer cell.
• PD-L2 showed a minor nuclear signal in MDA-MB-231 cells, but a positive PCC with LSDls111p ( Figure 2). This data indicates a nuclear presence of PD-L2 and a direct relationship between LSDlsll lp and PD-L2 in the nucleus of a cancer cell.
• Figure 3A depicts the FACS plots of inducible MCF7s stimulated with PMA or PMA and TGF- ⁇ that have been stained with PD-Ll .
• the mRNA of these cells was examined for PD-Ll, PD- L2 and CD44.
• PD-Ll mRNA expressed higher in MCF7 cells compared to PD-L2 ( Figure 3B).
• PD-L2 is expressed slightly higher in MDA-M B-231 cells compared to PD-L1.
• Both PD- Ll and PD-L2 induced upon stimulation of MCF7 cells.
• PD-L1 and PD-L2 expression is significantly higher in M DA-M B-231 cells compared with MCF7 cells.
• Table 5 depicts the stimulation of M DA-M B-231 breast cancer cells with Jurkat-T-cells.
• the frequency (%) of PD-L1+ cells is not altered by stimulation with IFNy (10 ⁇ g/mL), PMA (24 ng/mL) or PMA+IFNy.
• the frequency of PD-L1+ cells remains unchanged regardless of whether cells are stimulated for 24 or 48 hours.
• MFI Median Fluorescence Intensity
• Table 5 Stimulation of MDA-M B-231 breast cancer cells with Jurkat-T-cells (Frequency of parent, %).
• Table 6 Stimulation of MDA-M B-231 breast cancer cells with Jurkat-T-cells (Mean Fluorescence Intensity of Comp Pacific Blue-A PD-L1).
• cytoplasmic/surface expression of PD-Ll in both stimulated MCF7 cells and M DA-M B-231 cells Upon stimulation of the control MCF7 cells there is significant expression of nuclear PD-Ll as well as high levels of nuclear expression of PD-Ll. This is also evident in MDA-M B-231 cells. This, PD- Ll clearly has a strong nuclear presence in aggressive breast cancer cell lines. When treated with LI, L2 and L3, the expression of nuclear PD-Ll was significantly abrogated in both stimulated, mesenchymal MCF7 cells and M DA-M B-231 cells. This effect is lessened in the epithelial non- stimulated MCF7 cells.
• Figure 7 depicts the effect of treatment of a mouse M DA-MB-231 xenograft with either Abraxane (60 mg/kg) or Docetaxel (10 mg/kg) showing the volume of the tumors over time during treatment (Figure 7A).
• Surviving chemo-resistant MDA-MB-231 cells in either the Abraxane or Docetaxel treated cells exhibited a significantly increased fluorescent signal of LSDlsll lp [ Figure 7B (i)], EGFR [ Figure 7B (ii)] or SNAIL [ Figure 7B (Mi)] relative to xenograft M DA-M B-231 cells treated with vehicle alone when analysed using confocal laser scanning microscopy.
• Figure 8 depicts the effect of treatment with Abraxane, Phenelzine or a combination thereof on xenograft M DA-MB-231 cells.
• the effect of the combination treatment on the size and volume of the tumors for each of the group over time was assessed.
• Treatment with either Abraxane alone or in combination with Phenelzine resulted in a significant reduction of tumor volume.
• SNAIL and vimentin showing a significant drop as indicated.
• Cirulating tumor cells were isolated from meastatic breast cancer patient liquid biopsies. Representative confocal laser scanning microscopy images of isolated CTCs are presented in Figure 12. The marks detected include SNAIL, a transcription factor implicated in aggressive cancer with a mesenchymal state; vimentin ; cytokeratin ; LSDlsl llp; PD-L1 ; and PD- L2.
• LSDls111p and PD-L2 positive PCC was observed, which strongly indicates that these two markers interact in the nucleus.
• the patient samples all displayed a nuclear PD-L2 signal and the Fn/c showed either a bias towards the nucleus or a parity in signal intensity between the nucleus and cytoplasm.
• CTCs isolated from metastatic patient breast cancer liquid biopsies were labelled for cell surface vimentin, SNAIL and PD-L1 and analyzed using confocal laser scanning microscopy.
• PD-L1 clearly demonstrated a clear nuclear and cytoplasmic/cell surface signal (Figure 15).
• a general trend of increased nuclear and cytoplasmic signal intensities was observed in patients 1, 2, 3 and 4 whereas patients 5 and 6 displayed a reduction in both nuclear (TNFI) and cytoplasmic (TCFI) fluorescence after the first sample.
• TNFI nuclear
• TCFI cytoplasmic fluorescence after the first sample.
• the patient samples all displayed a nuclear PD-L1 signal and the Fn/c showed either a bias towards the nucleus or a parity in signal intensity between the nucleus and cytoplasm.
• SNAIL a target of LSDl regulation
• PD-L1 positive PCC was observed, which strongly indicates these two markers interact in the nucleus.
• CTCs isolated from two metastatic breast cancer patient liquid biopsies were treated with the LSDl catalytic inhibitors, Pargyline (Parg) or Phenelzine (Figure 16).
• the effects of the LSDl inhibitors was assessed by analyzing the expression of LSDls111p, cytokeratin (a marker for CTCs) and PD-L2 using confocal laser scanning microscopy.
• Both LSDl catalytic inhibitors, Pargyline and Phenelzine resulted in a significant knockdown of LSDls111p, PD-L2 and the CTC marker, cytokeratin. This strongly demonstrates that LSDl catalytic inhibitors can successfully abrogate LSDls111p and can knockdown PD-L2 and cytokeratin expression in patient CTCs.
• CTCs isolated from metastatic breast cancer patient liquid biopsies were treated with the LSDl catalytic inhibitor, Pargyline (Parg) ( Figure 17A).
• the effects of this inhibitor was determined by analyzing the expression of SNAIL (a target of LSDl regulation), vimentin and PD-Ll using confocal laser scanning microscopy. With the exception of one patient, Pargyline resulted in a significant knockdown of PD-Ll and SNAIL expression. This strongly suggests that LSDl catalytic inhibitors can abrogate PD-Ll and SNAIL expression in patient CTCs.
• Figures 18A and 18B show the effect of the cyclin-dependent kinase (CDK) inhibitors Palbociclib and Ribociclib, and the inhibitor of LSDl catalytic function, Phenelzine, and combinations thereof on isolated CTCs from metastatic breast cancer patient liquid biopsies using FACS.
• CDK cyclin-dependent kinase
• CD45-CK+ CD45-CK+ (Cytokeratin positive) CTCs.
• CD45-EpCAM + cells were detected.
• All test agents and combinations thereof inhibited PD-Ll nuclear (nPD-Ll) and surface/cytoplasmic (sPD-Ll) expression in CD45-CK+ CTCs ( Figure 18A).
• Ribociclib and the combination of Phenelzine and Ribociclib demonstrated the greatest inhibition of nuclear PD-Ll.
• CD45-EpCAM + CTCs were detected in PBMCs extracted from equivalent to 2 mL blood. 22.8% of CD45- cells were CD45-CK+ CTCs. Again, all test agents and combinations thereof inhibited PD-Ll nuclear expression in CD45-CK+ CTCs ( Figure 18B). However, only Ribociclib inhibited both nuclear and surface/cytoplasmic expression of PD-Ll.
• PKC- ⁇ inhibitors C27 or BIM significantly abrogated LSDl phosphorylation in both cell lines.
• this data indicates that a PKC, especially PKC- ⁇ , is involved in phosphorylation of LSDl at serine 111 and that such phosphorylation is important for nuclear localization.
• LSDl siRNA or the LSDl catalytic inhibitors NCD36 (2-[/V-(4- phenylbenzenecarbonyl)]amino-6-(trans-2-phenylcyclopropan-l-amino)-/V-(3- methylbenzyl)hexanamide hydrochloride; refer to EP 2927212 Al) or Pargyline (Parg) on the nuclear localization of LSDl and SNAIL was investigated in MCF7 cells treated with vehicle alone or stimulated with PMA for 60 hours, and M DA-M B-231 cells using confocal laser scanning microscopy. In stimulated MCF7 cells, nuclear SNAIL and LSDl was found to be increased ( Figures 28 and 29).
• MCF7 and M DA-MB-231 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in DM EM (Invitrogen, Life Technologies, Carlsbad, CA) supplemented with 10% FBS, 2mM L-glutamine, and 1% penicillin-streptomycin-neomycin. MCF7 cells were stimulated with 1.32 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St Louis, MO) or 5 ng/ml recombinant TGF- ⁇ (R&D Systems, M inneapolis, M N) for 60 h. For the co- culture assay M DA-MB-231 cells were stimulated with Jurkat T-cells at defined ratios.
• PMA phorbol 12-myristate 13-acetate
• TGF- ⁇ R&D Systems, M inneapolis, M N
• Cells were permeabilised by incubating with 2% Triton X-100 for 20 min. Cells were probed with either rabbit antibodies to LSDls111p (Merck ABE1462), EGFR (AB2430), MET (ab51067), PD-L1 (sc-50298), goat antibodies to PD-L1 (sc-19091), SC-14033), (sc-19096), mouse antibodies to Cytokeratin (M iltenyi 130-090-866), CSV (Abnova H00007431-M08), vimentin (SC-6260) followed by visualisation with secondary anti mouse Alexa-Fluor 568 (A10037), secondary anti rabbit Alexa-Fluor 488 (A21206) or secondary anti goat Alexa-Fluor 633 (A21082).
• Tumors were then finely minced using a surgical blade in a petri dish and incubated at 37°C for 1 hour with shaking in DM EM 2.5% FCS supplemented with collagenase type 4 (Worthington- Biochem, USA) (1 mg of collagenase / lg of tumor).
• Digested tumors were spun and resuspended in DM EM 2.5% FCS before being passed through a 0.2 ⁇ filter into a 50ml tube. Viable cells were then counted using trypan blue. A total number of 2 x 10 5 cells were stained for CD44-APC, CD24- PE and Hoescht.
• Flow cytometry acquisition was done using LSR II. Analysis of flow cytometry staining was done using the FlowJo software.
• LymphoprepTM (Catalogue #07801).
• the purified epithelial tumor cells were then extracted as a highly enriched population from the interface between the plasma and the buoyant density medium and were harvested in 20% FBS in PBS.
• Peripheral blood mononuclear cells (PBMCs) were isolated as well. PBMCs were stimulated with CD28 and P/I for 4 hours followed by co-culture with purified CD45 depleted cells for 12 hours with/without inhibitors of interest.
• Isolated PBMCs and CTCs were stained with CD45-APC, pan-cytokeratin (CK)-FITC, EpCAM-Percp-Cy5.5 and PD-L1-BV421 antibodies.
• Co-cultured M DA-MB-231 cells were stained with CD44, CD24 and PD-L1.
• Flow cytometric analysis was performed on single cell suspensions for using the BD LSR II Flow Cytometer.
• M DA-M B-231 or MCF7 cells were treated with one of LI, L2 or L3 and a control. Cells were then fixed with 3.7% formaldehyde and permeabilized with 2% Triton-X-100, then probed with a primary mouse antibody against LSDl or a primary rabbit antibody against PD-L1, EGFR or M ET, followed by visualization with a secondary goat antibody to mouse or rabbit immunoglobulins conjugated to Alexa-Fluor 488. Confocal laser scanning microscopy was used to measure the Fn/c (ratio of nuclear to cytoplasmic fluorescent intensity) ratio of LSDl with Fiji-imageJ or the total nuclear fluorescence of PD-L1, EGFR or M ET. Values are plotted with significant differences indicated using the Mann-Whitney T-test, with at least 20 cells counted per sample.
• TNFI Total nuclear fluorescence
• the average fluorescence signal intensity for the indicated pair of antibodies was plotted for each point on the line with SE. Signal was plotted to compare how the signals for each antibody varied compared to the opposite antibody. The PCC was determined for each plot profile, which indicates the strength of relation between the two fluorochrome signals for at least 20 individual cells ⁇ SE. Colours from representative images correspond to plot profiles. Immunofluorescence analysis of LSDl and SNAIL expression in MDA-MB-231 cells or MCF7 cells in response to treatment with LSDl inhibitors
• Test inhibitors include LSDl siRNA or LSDl catalytic inhibitors NCD36 (2-[/V-(4-phenylbenzenecarbonyl)]amino-6-(trans-2-phenylcyclopropan-l- amino)-/V-(3-methylbenzyl)hexanamide hydrochloride) or pargyline (Parg).
• TNFI values for M DA-M B-231, non-stimulated MCF7 and stimulated MCF7 cells were calculated. Data shown represent the mean ⁇ SE for at least 20 individual cells.
• Confocal laser scanning microscopy was performed on M DA-M B-231 cells treated with a LSDl peptide inhibitor, LI, L2 or L3, or vehicle alone. Cells were fixed with 3.7% formaldehyde and permeabilized with 2% Triton-X-100, and then probed with a primary mouse antibody to LSDl, followed by the corresponding secondary antibody conjugated to Alexa-Fluor 488. Confocal laser scanning microscopy was used to determine the Fn/c ratio of LSDl with image-J. Values are plotted with significant differences indicated. Data shown represent the mean ⁇ SE for at least 20 individual cells.
• LSDlsl llp The nuclear intensity of LSDlsl llp was measured using confocal laser scanning microscopy in xenografted M DA-M B-231 cells treated with vehicle alone or Abraxene (60mg/kg) or Docetaxel (lOmg/kg). Cells were fixed with 3.7% formaldehyde and permeabilized with 2% Triton- X-100, and then probed with a primary rabbit antibody to LSDls111p, followed by the
• TNFI Total nuclear fluorescence

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